Development and Characterization of Biomarker Assays for Alzheimer's Disease

Abstract

A doença de Alzheimer (DA) é uma doença cerebral neurodegenerativa e a forma mais comum de demência em todo o mundo, estando associada a altos custos sociais e financeiros. As duas principais caracteristicas patológicas são placas amilóides extracelulares contendo péptidos β-amiloides e emaranhados neurofibrilares compostos por formas hiperfosforiladas da proteína Tau.A patologia da DA começa décadas antes dos sintomas aparecerem, havendo por isso um grande interesse em desenvolver biomarcadores para um diagnóstico precoce da doença em indivíduos assintomáticos. Actualmente não existe cura para a DA e os medicamentos aprovados produzem apenas pequenas melhorias sintomáticas em alguns pacientes, mas não são capazes de impedir que a doença progrida. Portanto, existe uma enorme necessidade médica para o desenvolvimento de novas estratégias terapêuticas que atuem nos mecanismos patogénicos da DA com fortes propriedades modificadoras da doença que impeçam a progressão da doença em estágios pré-clínicos. Assim, os biomarcadores não só desempenham um papel no diagnóstico clínico, como também na compreensão da sequência de eventos moleculares no processo patogénico da DA e como ferramentas para monitorar os efeitos de novos candidatos a drogas visando esses diferentes mecanismos.In this project, we evaluated the interference of monoclonal Tau antibodies in the performance of the INNOTEST® P-Tau(181P) and T-Tau assays. Furthermore, we intended to develop more sensitive assays (MSD ELISA) to detect AD-related biomarkers. Additionally, we evaluated the performance of a newly synthesized lot of a multiplex Aβ MSD ELISA and compared the results to the performance of previous lot of the same assay. Finally, we tested the adsorption of Aβ to spinal catheters used for serial CSF collection.Our data showed interference of our tested antibodies in the T-Tau assay but not in the P-Tau assay, demonstrating that this assay can be used in Tau passive immunotherapy for longitudinal follow up of phospho-tau181 species in CSF. Our data showed that one phospho-tau antibody generated an immunoassay with increased sensitivity compared to the current gold standard immunoassay. We validated a newly synthesized 4 Plex Aβ lot for use in clinical studies and further used this lot to show a decrease in Aβ concentration in CSF samples that had passed through spinal catheters used for collection of lumbar CSF.Em conjunto, os nossos resultados mostram a importância de avaliar os métodos de recolha de LCR e o uso de ensaios de biomarcadores para orientar a decisão do tratamento ou projectar ensaios clínicos, além de permitir a interpretação inequívoca dos resultados de biomarcadores. Além disso, o desenvolvimento de novos imunoensaios que detectam Tau fosforilada ajudará a entender a dinâmica desta proteína durante o processo fisiopatológico da DA, e a distinguir de forma mais rigorosa pacientes controlo de pacientes com DA ou de indivíduos com outras tauopatias.

Alzheimer’s disease (AD) is a neurodegenerative brain disease and the most common form of dementia worldwide, being associated with high social and financial costs. The two main pathological hallmarks are extracellular amyloid plaques containing amyloid-β peptides, and neurofibrillary tangles composed of hyperphosphorylated forms of Tau protein. The pathology of AD begins decades before symptoms appear, hence there is a great interest in developing biomarkers for an early diagnosis of the disease on asymptomatic individuals. There is still no cure for AD and current medications produce only modest symptomatic improvements in some patients but are not able to stop the disease from progressing. Therefore, there is an enormous medical need for the development of novel therapeutic strategies that target the pathogenic mechanisms of AD with strong disease-modifying properties that halt disease progression at preclinical stages. Hence, biomarkers not only play a role in clinical diagnosis but also in the understanding of the sequence of molecular events in the pathogenic process of AD and as tools to monitor the effects of novel drug candidates targeting these different mechanisms. In this project, we evaluated the interference of monoclonal Tau antibodies in the performance of the INNOTEST® P-Tau(181P) and T-Tau assays. Furthermore, we intended to develop more sensitive assays (MSD ELISA) to detect AD-related biomarkers. Additionally, we evaluated the performance of a newly synthesized lot of a multiplex Aβ MSD ELISA and compared the results to the performance of previous lot of the same assay. Finally, we tested the adsorption of Aβ to spinal catheters used for serial CSF collection.Our data showed interference of our tested antibodies in the T-Tau assay but not in the P-Tau assay, demonstrating that this assay can be used in Tau passive immunotherapy for longitudinal follow up of phospho-tau181 species in CSF. Our data showed that one phospho-tau antibody generated an immunoassay with increased sensitivity compared to the current gold standard immunoassay. We validated a newly synthesized 4 Plex Aβ lot for use in clinical studies and further used this lot to show a decrease in Aβ concentration in CSF samples that had passed through spinal catheters used for collection of lumbar CSF.Taken together, our results show the importance of evaluating CSF collection methods and the use of biomarker assays for guiding treatment decision or designing clinical trials, and also for allowing unambiguous interpretation of biomarker results. Additionally, the development of new P-Tau immunoassays will help to understand tau dynamics during the AD pathophysiological process and to differentiate control patients from AD or other tauopathies.