Humanized Modification And Identification Of Murine Monoclonal Antibody Against SARS-CoV-2

In December 2019,the outbreak of COVID-19,caused by SARS-CoV-2,quickly spread to the world,seriously jeopardizing human health and global economic development.As the epidemic continues to recur,the situation is grim.The purpose of this project is to humanize the murine monoclonal antibody 20D8 against SARS-COV-2 obtained by hybridoma screening in the early stage,and functional identification of antibodies was performed,in order to lay a certain foundation for the development of SARS-CoV-2 therapeutic antibody drugs.In this project,total RNA 20D8 of hybridoma cells was firstly extracted and reversely transcribed into c DNA,and specific primers were designed to obtain the variable region sequences of heavy/light chain of murine monoclonal antibodies.Then,the variable region sequences of heavy/light chain of murine monoclonal antibody and the constant region sequences of humanized monoclonal antibody Ig G1/κwere splicted,and corresponding signal peptides and restriction sites were added,respectively,to construct recombinant plasmid into KS001 expression vector.The recombinant plasmid was constructed,and the chimeric antibody ch20D8 was expressed in Expi293F cells.The functional identification of antibodies was performed in vitro,and the pharmacodynamics of ch20D8 was evaluated in K18-h ACE2transgenic mice.The results showed that the affinity of ch20D8 hardly decreased after chimeric modification.The EC₅₀ of ch20D8 against RBD（prototype strain）was 2.848 ng/m L,the IC₅₀ of ch20D8 against RBD（prototype strain）and ACE2 was 156.8 ng/m L,and the IC₅₀of ch20D8 against pseudovirus（prototype strain）was 1.504 ng/m L.It also had strong binding,blocking and neutralizing activities against other mutant strains.The results of pharmacodynamic evaluation in mice showed that ch20D8 had a good protective effect on K18-h ACE2 mice infected with live virus（B.1.617.2 strain）.The above results proved the correctness of the variable region sequence of the obtained murine monoclonal antibody and the chimeric modification of murine monoclonal antibody was successful.In order to further reduce the components of murine monoclonal antibody,CDR transplantation technique and reverse-mutation technique were used to humanize murine monoclonal antibody 20D8,and functional identification of antibodies was performed.First,a humanized combined mutation Fab library was constructed and screened to obtain Fab clones with high affinity and few reverting mutation sites.Then,the Fab clones were inserted into pc DNA3.4 vector containing the antibody constant region（Ig G1/κ）sequence to construct the complete heavy/light chain recombinant plasmid.The plasmid was transfected into Expi293F cells to express humanized monoclonal antibody h20D8.Then a series of identification of h20D8,including physicochemical and biological activity identification.Physicochemical identification results showed that the heavy/light chain size of h20D8 was basically consistent with the theoretical molecular weight,with high purity and isoelectric point of 7.928～8.716.Biological activity identification results showed that the EC₅₀ of h20D8 against RBD（prototype strain）was 3.641 ng/m L,the IC₅₀ of h20D8 against RBD（prototype strain）and ACE2 was 227.6 ng/m L,and the IC₅₀ of neutralizing activity against pseudovirus and live virus（prototype strain）were 2.874 ng/m L and 14.46 ng/m L,respectively,and they also showed strong binding,blocking and neutralizing activity against other mutant strains.Compared with ch20D8,the binding activity,blocking activity and neutralizing activity of h20D8 almost did not decrease,and the humanized modification was successful.In conclusion,the variable region sequence of murine monoclonal antibody 20D8 was obtained in this project,and the humanized modification was successfully carried out.The affinity and biological activity of the modified h20D8 monoclonal antibody almost did not decrease,and it had strong neutralizing activity against the prototype strain,B.1.1.7,B.1.351,P.1 and B.1.617.2 mutants.This laid a foundation for the subsequent development of broad-spectrum and highly effective SARS-CoV-2 therapeutic antibody drugs.