Development and Characterization of Mouse-Specific Anti-Tau Monoclonal Antibodies: Relevance for Analysis of Murine Tau in Cerebrospinal Fluid.

BACKGROUND: Clearance of tau seeds by immunization with tau antibodies is currently evaluated as therapeutic strategy to block the spreading of tau pathology in Alzheimer's disease and other tauopathies. Preclinical evaluation of passive immunotherapy is performed in different cellular culture systems and in wild-type and human tau transgenic mouse models. Depending on the preclinical model used, tau seeds or induced aggregates can either be of mouse, human or mixed origin. OBJECTIVE: We aimed to develop human and mouse tau-specific antibodies to discriminate between the endogenous tau and the introduced form in preclinical models. METHODS: Using hybridoma technology, we developed human and mouse tau-specific antibodies that were then used to develop several assays to specifically detect mouse tau. RESULTS: Four antibodies, mTau3, mTau5, mTau8, and mTau9, with a high degree of specificity for mouse tau were identified. Additionally, their potential application in highly sensitive immunoassays to measure tau in mouse brain homogenate and cerebrospinal fluid is illustrated, as well as their application for specific endogenous mouse tau aggregation detection. CONCLUSION: The antibodies reported here can be very important tools to better interpret the results obtained from different model systems as well as to study the role of endogenous tau in tau aggregation and pathology observed in the diverse mouse models available.

Passive immunotherapy with a novel antibody against 3pE-modified Aß demonstrates potential for enhanced efficacy and favorable safety in combination with BACE inhibitor treatment in plaque-depositing mice.

The imbalance between production and clearance of amyloid ß (Aß) peptides and their resulting accumulation in the brain is an early and crucial step in the pathogenesis of Alzheimer's disease (AD). Therefore, Aß is strongly positioned as a promising and extensively validated therapeutic target for AD. Investigational disease-modifying approaches aiming at reducing cerebral Aß concentrations include prevention of de novo production of Aß through inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and clearance of Aß deposits via passive Aß immunotherapy. We have developed a novel, high affinity antibody against Aß peptides bearing a pyroglutamate residue at amino acid position 3 (3pE), an Aß species abundantly present in plaque deposits in AD brains. Here, we describe the preclinical characterization of this antibody, and demonstrate a significant reduction in amyloid burden in the absence of microhemorrhages in different mouse models with established plaque deposition. Moreover, we combined antibody treatment with chronic BACE1 inhibitor treatment and demonstrate significant clearance of pre-existing amyloid deposits in transgenic mouse brain, without induction of microhemorrhages and other histopathological findings. Together, these data confirm significant potential for the 3pE-specific antibody to be developed as a passive immunotherapy approach that balances efficacy and safety. Moreover, our studies suggest further enhanced treatment efficacy and favorable safety after combination of the 3pE-specific antibody with BACE1 inhibitor treatment.

Discovery and Functional Characterization of hPT3, a Humanized Anti-Phospho Tau Selective Monoclonal Antibody.

BACKGROUND: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. OBJECTIVE: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. METHODS: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. RESULTS AND CONCLUSION: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.

Anti-Tau Monoclonal Antibodies Derived from Soluble and Filamentous Tau Show Diverse Functional Properties in vitro and in vivo.

The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.

Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti-tau antibody AT8.

Microtubule-associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF-tau). AT8 is a PHF-tau-specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30-fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF-tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202-209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR-L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody.

Macrocyclic BACE inhibitors: Optimization of a micromolar hit to nanomolar leads.

We have identified macrocyclic inhibitors of the aspartic protease BACE, implicated in the etiology of Alzheimer's disease. An X-ray structure of screening hit 1 in the BACE active site revealed a hairpin conformation suggesting that constrained macrocyclic derivatives may also bind there. Several of the analogs we prepared were >100x more potent than 1, such as 7 (5 nM K(i)).

Reduction of Abeta levels in the Sprague Dawley rat after oral administration of the functional gamma-secretase inhibitor, DAPT: a novel non-transgenic model for Abeta production inhibitors.

Considerable effort has been made to develop drugs that delay or prevent neurodegeneration. These include inhibitors of Abeta-generating proteases for the treatment of Alzheimer's disease. Testing the amyloid hypothesis in vivo requires molecules that are capable of entering the CNS and that produce a substantial reduction in brain Abeta levels. Plaque-developing APP transgenic mice are currently widely used as an in vivo model of choice as these animals produce readily measurable amounts of human Abeta. They are very useful in the testing of a variety of amyloid-lowering approaches but their use for compound screening is often limited by their cost. Transgenic animals also require extensive, time-consuming breeding programs and can show high inter-animal differences in the expression level of the transgene. Hence, we considered it important to develop and characterize a new and simple non-transgenic animal model for testing Abeta modulation. For this purpose, Wild-type adult Sprague Dawley rats were treated with DAPT, a functional gamma-secretase inhibitor, and the Abeta40 and Abeta42 levels in brain-tissue and body fluids were assessed. We showed that DAPT, given orally, significantly lowered Abeta40 and Abeta42 peptide levels in brain extract, CSF, and the plasma dose- and time-dependently. We can conclude that our data establish the usefulness of the wild-type rat model for testing small-molecule inhibitors of Abeta production.

Development of a specific ELISA for the quantitative study of amino-terminally truncated beta-amyloid peptides.

Clinical studies for disease modifying drugs in Alzheimer's disease are in real need for a sensitive biochemical diagnostic and therapeutic marker. Encouraging results have been obtained by measuring levels of pathology related proteins such as amyloid beta (Abeta) peptides and tau proteins in cerebrospinal fluid (CSF) and plasma of patients. We and other research groups have shown that truncated Abeta11-40 and Abeta11-42 is also a potential marker and that it is produced and deposited in the brains of patients and transgenic mouse models. Because of a lack of quantitative methods for specific measurement of the truncated Abeta it has not been possible to evaluate the true value of this potential biomarker. To overcome these limitations we developed a novel monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to measure Abeta11-40 and Abeta11-42 levels in biological materials. For this purpose monoclonal antibodies were produced that react specifically with amyloid precursor protein (APP) products generated by cleavage at position 11 of the Abeta sequence. The assay has the sensitivity, selectivity and dynamic range to allow specific, direct quantitation of truncated Abeta11 peptides in cell culture medium, cerebrospinal fluid and brain tissue extracts.