| Background Influenza A virus(IAV)is a respiratory pathogen that can infect wild birds,poultry and humans,posing a significant threat to the global economy and public health security.To date,subtypes H5 and H7 highly pathogenic influenza have resulted in more than 2,600 human cases and more than1,000 deaths worldwide.With sporadic cases of human infections of avian influenza subtypes H5 and H7 and no effective vaccine immunization against subtypes H5 and H7,there is an urgent need to develop effective therapeutic drugs.Monoclonal antibodies have the advantages of clear targeting,high purity,and mass production,and are an important tool for the prevention and treatment of viral infectious diseases such as influenza.Bispecific antibodies targeting multiple antigens or antigenic epitopes play a synergistic role that is more advantageous than monoclonal antibodies,allowing them to cope with different subtypes of influenza virus strains,enhance antibody neutralization activity,and more effectively inhibit the occurrence of virus escape.ObjectiveTo construct bispecific neutralizing antibodies with therapeutic value against H5 subtype and H7 subtype,and to investigate the recognition epitopes and mechanism of action of bispecific antibodies.MethodSix H5/H7 bispecific antibodies were constructed by two design schemes,DVD-Ig and Ig G-sc Fv,using H5 subtype monoclonal antibody 3CF5 and H7 subtype monoclonal antibody 41B5 as parental antibodies.Expression verification of the antibodies in 293 F cells,binding assay and affinity activity verification with influenza virus HA protein and neutralization assay with influenza virus;screening of virus escape strains under antibody stress as well as point mutation of HA protein binding sites using yeast surface display system to compare bispecific antibodies and parental antibodies in terms of their ability to tolerate virus escape and binding epitopes;ADCC experiments were performed to verify the mechanism of action of the bispecific antibodies.Results(1)Six bispecific antibodies named bs5701,bs5702,bs5703,bs5704,bs5705 and bs5706 were successfully constructed,all of which were expressed in mammalian cells and their transfected supernatants had the ability to bind H5/H7 subtype HA protein and neutralize H5/H7 subtype fluorescent virus;(2)two bispecific antibodies numbered bs5704 and bs5706 had comparable or better binding ability to H5 and H7 subtype HA compared to the parental antibody,and the other bispecific antibody bs5703 changed the binding mode and was able to bind HA stem protein;(3)bs5704 and bs5706 exhibited broad-spectrum neutralizing activity against H5 subtype influenza virus and H7 subtype virus;(4)compared to the parental bs5704 and bs5706 showed higher affinity activity for A/Sichuan/26221/2014(H5N6)HA and A/Anhui/1/2013(H7N9)HA compared with parental antibodies;(5)virus escape assay under antibody stress showed that bispecific antibodies were more resistant to virus escape than parental antibodies,and after three generations of chicken embryo screening bs5704 and bs5704 and bs5706 were not screened for H7N9 virus escape,while the parental antibody showed virus escape;(6)the bispecific antibody retained the ability of the parental antibody 3CF5 to activate effector cells to induce ADCC effects on H5 subtype antigens.ConclusionThe bispecific antibodies bs5704 and bs5706 possess simultaneous broad-spectrum efficient neutralizing activity against H5/H7 subtype influenza viruses,synergistically enhance each other’s ability to tolerate viral escape,and retain the ADCC effect of the parental antibody,which can be used as therapeutic antibodies against H5 and H7 subtypes and have greater value for clinical research applications. |
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