Application of anti-amyloid beta immunization using encapsulated cell technology in mouse models of Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia in the elderly. AD is characterized by the deposition of two aggregated proteins: Amyloid beta (Aß) and hyperphosphorylated tau. Accumulations of these proteins are thought to be the signature of a pathogenic process causing the loss of neurons and synaptic connections associated with severe cognitive impairments in AD patients. Other important factors are involved in the neurodegeneration occurring in AD, such as neuroinflammation. In the present thesis work, we are investigating the effects of passive anti-Aß immunization. Passive anti-Aß immunization consists in the delivery of antibodies targeting Aß, with the aim to prevent its accumulation and promote the removal of pathological amyloid species. Although the treatment has been shown to clear amyloid pathology in the brain of AD patients, it has so far failed to prevent or slow down cognitive decline. Hence, it is important to determine the effects of anti-Aß immunization when administered either before or during Aß deposition, and explore how the treatment affects the Alzheimerâs manifestations in mouse models combining Aß and tau pathologies. In order to continuously deliver antibodies in AD mouse models, we used encapsulated cell technology (ECT) to implant myoblasts genetically engineered and secrete the recombinant mAb11 mouse IgG2a antibody directed against aggregated forms of Aß. Using this approach, we investigated the following three topics of research:

We examined the effect of a preventive administration of anti-Aß antibodies in a slowly progressing mouse model with both Aß and tau (P301L) pathologies. We demonstrated that delivery of anti-Aß antibodies before the onset of the disease dramatically reduces Aß pathology. We also found that the treatment decreases hyperphosphorylated tau accumulation and recruits microglial around Aß plaques.
We next assessed the effects of passive anti-Aß immunization in another AD mouse model overexpressing both Aß and wild-type (WT) tau. In this second study, we administered the anti-Aß antibodies when both Aß and tau pathologies were already established. In the hippocampal formation, we showed that WT tau overexpression might affect the microglial response to ongoing pathology by reducing the tight interaction of these cells with the amyloid plaques. This effect, however, is counteracted by passive anti-Aß immunization, which was found to redirect microglia towards Aß deposits. In addition, passive anti-Aß immunization decreases tau spreading throughout the hippocampus, but fails to prevent neither tau phosphorylation, nor the hippocampal degeneration induced by local tau overexpression.
In parallel, we developed a human myoblast cell line secreting anti-Aß antibodies for translation of our ECT towards human application. Using C2C12 mouse myoblasts, our flat-sheet device allowed for long-term continuous antibody delivery in the plasma of subcutaneously implanted mice. We were able to generate antibody-secreting human cells using the immortalized C25 cell It was possible to differentiate these cells into myotubes and adapt them to long-term survival inside the capsules maintained in vitro. However, when implanted in the subcutaneous tissue of immunocompromised mice, myoblasts survived in the ECT device as a thin layer of cells and were not able to secrete recombinant antibodies in sufficient amounts to be considered for therapeutic purpose.

A novel encapsulation device for peripheral implantation of antibody-secreting cells

Aurélien Lathuilière

Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly. As populations are aging in most parts of the world, the number of people with Alzheimer’s disease is expected to dramatically increase. This devastating neurodegenerative disorder remains the only major source of mortality without any effective disease-modifying treatment. Nevertheless, the progress made in understanding underlying disease mechanisms linked to amyloid and tau pathologies, led to the development of novel therapeutic strategies. The most clinically advanced experimental treatment is based on monoclonal antibodies (passive immunization) directed against the amyloid β peptide, a critical contributor to the neuropathology of Alzheimer’s disease. Although immunotherapy is able to clear cerebral amyloid deposits in patients, it has so far failed to slow down the loss of cognitive functions. In order to potentiate therapeutic effects, it may be critical to initiate the treatment before the first cognitive symptoms, and therefore chronically administer antibodies over years, possibly decades. This prompts the development of alternative strategies for the long-term delivery of therapeutic antibodies. To address this question, we have investigated an innovative approach based on a biologically active subcutaneous device encapsulating cells genetically engineered for the production of recombinant antibodies. In order to demonstrate that encapsulated cell technology can achieve peripheral continuous delivery of antibodies and lead to significant antibody exposure inside the brain, we rationally optimized parameters that are crucial to the rate of antibody secretion from such a cellular implant. In the first part of the thesis, we explored the utilization of lentiviral vectors for the genetic engineering of antibody secreting cell lines. We developed a dual vector strategy to significantly improve the efficiency of stable cell line generation. Moreover, the lentiviral transduction of C2C12 murine myoblasts allowed for the isolation of individual clones demonstrating high cell specific productivity, thus validating the use of C2C12 as a preclinical surrogate cell line for allogeneic implantation in disease mouse models. In the second part of the present work, we engineered a novel cell encapsulation device specially designed for subcutaneous implantation.We implemented a highly reproducible and scalable process to assemble devices supporting the implantation of large quantities of cells. Moreover, the in vivo optimization of initial cell seeding parameters enabled encapsulated cells to survive for months at high density inside the device. The final part of the present work assessed therapeutic efficacy of the newly developed cell encapsulation system for peripheral immunization against brain amyloid pathology. Devices secreting anti-Aβ antibodies were subcutaneously implanted in two transgenic mouse models of Alzheimer’s disease. Antibody levels in the mouse plasma reached 40 μg/ ml for several months, demonstrating long-term implant efficacy. Furthermore, we could demonstrate immunodecoration of amyloid plaques inside the brain with the delivered antibody, demonstrating penetration into the central nervous system. Remarkably, in the two mouse models of Alzheimer’s disease, brain amyloidosis was significantly reduced in the treated animals. Overall, this thesis work shows that peripheral implantation of encapsulated antibody-secreting myoblast cells is an effective strategy for passive immunization, particularly in the challenging context of delivering therapeutic amounts of antibodies to the central nervous system. Indeed, we demonstrate that anti-Aβ antibodies administered by subcutaneous bioactive implants can significantly decrease amyloid burden in mouse models of Alzheimer’s neuropathology.

EPFL2013

A viral vector based in vivo model of tauopathy to explore therapeutic strategies against tau pathology

Sameer Mohammad Khaleel Nazeeruddin

Tauopathies are neurodegenerative diseases whose common pathological feature is the intraneuronal accumulation of tau aggregates. Tau protein has various roles in the neuron, among which stabilization of microtubules (MT), regulation of synaptic activity or axonal transport. Subsequently, tau pathology exerts toxicity through mechanisms that likely involve the loss of normal function or the gain of toxic functions. In tauopathies, tau undergoes post-translational modifications (PTM), which are thought to promote the detachment from the MT, change its subcellular localization, and induce conformational changes that favor its assembly with other tau monomers, forming oligomers and fibrils. Furthermore, both normal and pathological forms of tau can be transferred from neuron to neuron. This process may have an important role in disease progression, by propagating the pathology along synaptically connected brain regions, which reflects the evolution of the symptoms. Therefore, the aggregation of tau protein and its propagation are promising targets for potential treatments. In the present thesis, our objective was to assess the efficacy of two treatments using a mouse model of tauopathy based on local overexpression of the 4R0N human tau (hTau) wild-type (WT) induced by intracerebral injection of an adeno-associated viral (AAV) vector. In the first part of the thesis, we overexpressed tau in the entorhinal cortex (EC) of WT mice to induce pathology and assess the effects of aggregation inhibitor compounds orally administered. Although we did not observe any effect on markers of tau aggregation, likely because the induced pathology was still at an early stage, the treatment was found to enhance phosphorylation of the Ser202/Thr205 residues in the EC and ipsilateral hippocampus (HPC). Additionally, we measured an increase in the density of microglial cells in the HPC, and significantly decreased levels of total hTau in the cerebrospinal fluid of the treated mice. These findings suggest a potential positive effect of this treatment on the clearance of hTau. In the second part of the thesis, we unilaterally overexpressed hTau in the mouse CA3 HPC and used the connectivity between both hemispheres to quantify the propagation of tau from neuron-to-neuron to the contralateral HPC. We found neurons labelled for hTau in the contralateral HPC, the number of which was increased in 5xFAD mice carrying the amyloid pathology. Next, to induce an antibody response against pathological tau, we subcutaneously injected a vaccine against the phospho-Ser396/404 tau epitope. The vaccine induced a potent antibody response in WT mice, but antibody titers remained low in the 5xFAD mice. In the treated WT mice, immunohistochemistry showed a decreased PHF-1 immunoreactivity in the HPC, indicating that the generated antibodies indeed recognized the phospho-Ser396/404 epitope. Remarkably, the vaccine decreased the number of neurons positive for hTau in the contralateral HPC, suggesting an effect of immunotherapy on tau propagation. Furthermore, the vaccine also showed potential neuroprotective effects, by decreasing the level of neurodegeneration in the dentate gyrus of treated mice as a function of the anti-phospho-tau antibody titer. In this work, using adapted AAV8-based models of local tau overexpression in the mouse brain, we reveal the effects of two treatments on different aspects of tau pathology related to the progression of the symptoms in tauopathies.

EPFL2019

Anti-Aβ antibodies bound to neuritic plaques enhance microglia activity and mitigate tau pathology

Philippe Colin, Vanessa Laversenne, Bernard Schneider

The brain pathology of Alzheimer’s disease (AD) is characterized by the misfolding and aggregation of both the amyloid beta (Aβ) peptide and hyperphosphorylated forms of the tau protein. Initial Aβ deposition is considered to trigger a sequence of deleterious events contributing to tau pathology, neuroinflammation and ultimately causing the loss of synapses and neurons. To assess the effect of anti-Aβ immunization in this context, we generated a mouse model by overexpressing the human tau protein in the hippocampus of 5xFAD mice. Aβ plaque deposition combined with human tau overexpression leads to an array of pathological manifestations including the formation of tau-positive dystrophic neurites and accumulation of hyperphosphorylated tau at the level of neuritic plaques. Remarkably, the presence of human tau reduces microglial clustering in proximity to the Aβ plaques, which may affect the barrier role of microglia. In this mouse model, continuous administration of anti-Aβ antibodies enhances the clustering of microglial cells even in the presence of tau. Anti-Aβ immunization increases plaque compaction, reduces the spread of tau in the hippocampal formation and prevents the formation of tau-positive dystrophic neurites. However, the treatment does not significantly reduce tau-induced neurodegeneration in the dentate gyrus. These results highlight that anti-Aβ immunization is able to enhance microglial activity around neuritic plaques, mitigating part of the tau-induced pathological manifestations.