Background:Amyloid-β(Aβ)immunotherapy is a promising therapeutic strategy in the fght against Alzheimer’s disease(AD).A number of monoclonal antibodies have entered clinical trials for AD.Some of them have failed d...Background:Amyloid-β(Aβ)immunotherapy is a promising therapeutic strategy in the fght 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 efcacy or side-efects,two antibodies are currently in phase 3,and one has been approved by FDA.The soluble intermediate aggregated species of Aβ,termed oligomers and protofbrils,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.Methods:We designed and recombinantly produced a hexavalent antibody based on mAb158,an Aβprotofbrilselective 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 Aβprotofbrils.Diferent ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to Aβaggregates of diferent sizes.Finally,the ability of the antibodies to protect cells from Aβ-induced efects was evaluated by MTT assay.Results:Using real-time interaction analysis with LigandTracer,the hexavalent design promoted a 40-times enhanced binding with avidity to protofbrils,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 fbrils.The hexavalent antibody also reduced cell death induced by a mixture of soluble Aβaggregates.Conclusion:We provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of Aβaggregates.This approach should be general and work for any aggregated protein or repetitive target.展开更多
基金This work was supported by grants from Swedish Research Council,Hedlunds stiftelse,Åke Wibergs stiftelse,Åhlen-stiftelsen,Jeanssons stiftelser,Magnus Bergvalls stiftelse,Vinnova and Alzheimerfonden。
文摘Background:Amyloid-β(Aβ)immunotherapy is a promising therapeutic strategy in the fght 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 efcacy or side-efects,two antibodies are currently in phase 3,and one has been approved by FDA.The soluble intermediate aggregated species of Aβ,termed oligomers and protofbrils,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.Methods:We designed and recombinantly produced a hexavalent antibody based on mAb158,an Aβprotofbrilselective 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 Aβprotofbrils.Diferent ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to Aβaggregates of diferent sizes.Finally,the ability of the antibodies to protect cells from Aβ-induced efects was evaluated by MTT assay.Results:Using real-time interaction analysis with LigandTracer,the hexavalent design promoted a 40-times enhanced binding with avidity to protofbrils,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 fbrils.The hexavalent antibody also reduced cell death induced by a mixture of soluble Aβaggregates.Conclusion:We provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of Aβaggregates.This approach should be general and work for any aggregated protein or repetitive target.
