Engineering a Dimeric Single-Domain Antibody for Improved Detection and neutralization of Amyloid-β oligomers

Soluble A{beta} oligomers are regarded as major neurotoxic agents in Alzheimers disease. Several monoclonal antibodies have been developed to target A{beta} oligomers, but most of them show limited specificity binding also to monomers and fibrils. To generate an antibody with high specificity for the oligomers, we aimed to increase the efficiency and sensitivity of a humanized A{beta}-oligomer-specific single domain antibody, called DesAb-O. We engineered a dimeric DesAb-O variant, DiDesAb-O, which showed a significantly improved binding properties for A{beta} oligomers as compared to the monomeric sdAb. Furthermore, DiDesAb-O detected A{beta}42 oligomers in cells, prevented their binding to cell membranes and the A{beta}42 oligomers-induced neurotoxicity from both synthetic A{beta}42 samples and cerebrospinal fluid of Alzheimers patients at lower concentrations compared to DesAb-O. Overall, our findings indicate that the rational engineering of dimeric sdAb variants is an effective strategy to improve their binding properties offering new opportunities for the development of clinical molecules in the early diagnosis and cure of Alzheimers disease. Significance StatementProtein aggregates, characterized by high structural heterogeneity, are central to numerous neurodegenerative diseases, yet the development of aggregate-specific molecular probes and therapeutic agents remains a significant challenge. We present a novel approach to enhance the binding of single-domain antibodies by engineering multivalent, specifically dimeric, molecules to exploit the avidity effect. As a proof of concept, we developed a dimeric version of a previously studied single-domain antibody named DesAb-O, which exhibits enhanced binding sensitivity and specificity to toxic amyloid-{beta} oligomers and greater efficacy in inhibiting their toxicity. These findings provide a robust foundation for creating next-generation antibody fragments with enhanced binding to heterogeneous protein aggregates, opening new avenues for innovative diagnostic tools and therapeutic strategies in neurodegenerative disease research and treatment.