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Background: Patients with Alzheimer’s disease (AD) have considerably increased globally as a result of population aging, placing a significant burden on the global economy and the medical system. The outcome of clinical trials for AD immunotherapy that solely targeted amyloid-β (Aβ) or phosphorylated tau protein (p-Tau) was unsatisfactory. Therefore, blocking both Aβ and p-Tau’s pathological processes simultaneously while also preventing their interaction may be the key to developing an effective AD therapy. Objective: To develop a novel immunotherapy based on bispecific tandem scFv (TaFv) against AD. Methods: Bispecific single-chain antibody that targets both Aβ and p-Tau were obtained using E. coli expression system. Biological ability of TaFvs were determined by ELISA, SDS-PAGE, and immunohistochemical assay. Recombinant adeno-associated virus 9 (rAAV9) were packaged to create TaFv. The in vivo activity of rAAV9 were detected in mouse, using biophotonic imaging and frozen section methods. Results: The outcomes demonstrated that both Aβ and p-Tau had a high affinity for the bispecific TaFv. Additionally, it can bind to the amyloid plaques and neuronal tangles in the brain slices of an AD mouse model. Moreover, the rAAV9 could infect neuronal cells, transverse the blood-brain barrier, and express TaFv in the mouse brain. Conclusion: This novel immunotherapy offers a fresh concept for the immunotherapy of AD and successfully delivers the double target antibody into the brain, acting on both pathogenic substances Aβ and p-Tau.

Disease-modifying passive immunotherapies focusing on removal of abnormal phosphorylated Tau (p-Tau) constitute promising treatments for Alzheimer's disease (AD). Although several prior immunotherapies targeting p-Tau appear to be beneficial against AD, they have limitations such as the low blood-brain barrier (BBB) penetration rate, short half-life of antibodies, and the likelihood of inflammation. To address these issues, we designed a novel immunotherapy for AD. To this end, a single chain antibody (scFv) targeting p-Tau was generated, and a recombinant adeno-associated virus that can cross the BBB (rAAV/BBB) was used as a vector to express scFv for at least 22 weeks in the mouse brain. Results showed that the scFv constructed in this study had a high affinity to p-Tau and could bind to neuronal tangles in the section of brains of AD model mice. Moreover, the rAAV/BBB could cross the BBB, infect neuronal cells, and express scFv. This novel immunotherapy could effectively deliver scFv into the brain and resulted in a continuous expression of scFv in vivo, suggesting its potential for the treatment of AD.