Abstract
BackgroundAmyloid-β (Aβ) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer’s disease (AD). A number of monoclonal antibodies have entered clinical trials for AD. Some of them have failed due to the lack of efficacy or side-effects, two antibodies are currently in phase 3, and one has been approved by FDA. The soluble intermediate aggregated species of Aβ, termed oligomers and protofibrils, are believed to be key pathogenic forms, responsible for synaptic and neuronal degeneration in AD. Therefore, antibodies that can strongly and selectively bind to these soluble intermediate aggregates are of great diagnostic and therapeutic interest.MethodsWe designed and recombinantly produced a hexavalent antibody based on mAb158, an Aβ protofibril-selective antibody. The humanized version of mAb158, lecanemab (BAN2401), is currently in phase 3 clinical trials for the treatment of AD. The new designs involved recombinantly fusing single-chain fragment variables to the N-terminal ends of mAb158 antibody. Real-time interaction analysis with LigandTracer and surface plasmon resonance were used to evaluate the kinetic binding properties of the generated antibodies to Aβ protofibrils. Different ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to Aβ aggregates of different sizes. Finally, the ability of the antibodies to protect cells from Aβ-induced effects was evaluated by MTT assay.ResultsUsing real-time interaction analysis with LigandTracer, the hexavalent design promoted a 40-times enhanced binding with avidity to protofibrils, and most of the added binding strength was attributed to the reduced rate of dissociation. Furthermore, ELISA experiments demonstrated that the hexavalent design also had strong binding to small oligomers, while retaining weak and intermediate binding to monomers and insoluble fibrils. The hexavalent antibody also reduced cell death induced by a mixture of soluble Aβ aggregates.ConclusionWe provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of Aβ aggregates. This approach should be general and work for any aggregated protein or repetitive target.
Highlights
Amyloid-β (Aβ) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer’s disease (AD)
A dual variable domain (DVD) antibody was designed, where an additional heavy chain variable domain was attached to the N-terminal end of the heavy chain and an additional light chain variable domain was attached to the N-terminal end of the light chain (Fig. 1b)
The slightly lower inflection temperatures detected for Tetra- and Hexa-RmAb158 antibodies can be attributed to the presence of Single chain fragment variable (scFv) on the N-terminal ends of these antibodies, as scFvs are more susceptible to thermal shifts than full IgG antibodies
Summary
Amyloid-β (Aβ) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer’s disease (AD). Antibodies that can strongly and selectively bind to these soluble intermediate aggregates are of great diagnostic and therapeutic interest. As the number of connected binding sites on one target increases, the rate of dissociation from the target will decrease. This effect is called avidity, which is defined as the unified strength of multiple interactions between an antibody and its target [1, 2]. IgM antibodies with 10 binding sites can have higher avidity than IgG antibodies, when multiple epitopes on a target are bound simultaneously. It has been shown that antibodies modified into a tetravalent form have higher avidity than bivalent antibodies [3]
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