Preparation And Biological Activity Study On Anti-SARS-CoV-2 Bispecific Single-Chain Antibody

COVID-19 has leaded to disastrous consequences in more than 200 countries and regions and is now the biggest public threat in the world.COVID-19 is a severe and highly infectious zoonotic disease caused by a novel coronavirus named SARS-Co V-2.The constantly mutating SARS-Co V-2 has been infected an increasing number of people,despite the availability of multiple approved COVID-19 vaccines worldwide,hence the safe and effective treatment and prevention measures for COVID-19 are urgently needed.SARS-Co V-2 spike protein（S）receptor binding domain（RBD）,a key target of binding receptor cells,has been the main target of antibody drug development.At present,there are many studies on monoclonal antibodies against SARS-COV-2,mainly using phage display,flow cytometry and other technologies to obtain neutralizing antibodies.Single chain antibody fragment（sc Fv）,as a representative drug of small molecule antibody,has the advantages of short development time,low production cost,easy genetic engineering operation and short half-life.Bispecific antibody（Bs Ab）has two different antigen-binding arms,which combines the characteristics of monomolecular strategy and cocktail therapy,and can simultaneously bind two different antigens or epitopes,which has incomparable advantages over monoclonal antibody.At present,there are little researches on anti-SARS-Co V-2 sc Fv and Bs Ab.Therefore,we studied the sc Fv and bispecific single chain antibody（Bsc Ab）against SARS-COV-2 RBD.Objective:To construct antibodies in sc Fv and Bsc Ab format on the basis of Nabs COVA1-16,COVA2-29 and CR3022,which are derived from donors who had recovered from COVID-19 or SARS Co V.To optimize the production process of each antibody and then evaluate their binding activity to SARS-Co V-2 RBD and neutralizing activity to SARS-Co V-2.To evaluate the synergistic effect of S3022 with other sc Fvs as a component of the antibody cocktail or a subunit of a bispecific single chain antibody.To analyze the different epitopes of each sc Fv binding to SARS-COV-2 RBD using bioinformatics methods and verify the predicted results using competitive ELISA.Contents:Using a prokaryotic expression system,sc Fv S1-16,S2-29,S3022 and their fusion proteins 16-29 and 16-3022 in Bsc Ab format were developed based on the Nabs COVA1-16,COVA2-29 and CR3022,which are derived from donors who had recovered from COVID-19 or SARS Co V.The induction,purification and renaturation technology of the antibody molecules was optimized.The binding activity and neutralizing activity of the antibodies or antibody cocktail to SARS-Co V-2 were evaluated.Using bioinformatics methods,we confirmed that SARS-Co V RBD targeting sc Fv S3022 could play a synergistic role with other SARS-Co V-2 RBD targeting antibodies to enhance neutralization activity.Method:Three sc Fvs and two Bsc Abs against SARS-Co V-2 were expressed in E.coli BL21.After inclusion body dissolution,the antibodies were purified on Ni-NTA column and obtained by dialysis renaturation.ELISA and Western blot were used to identify the binding activity of five antibodies to SARS-COV-2 RBD.The cytotoxicity of five antibodies was determined by MTT assay.Pseudotyped virus neutralization test was used to evaluate the neutralizing activity of five antibodies and an antibody cocktail against SARS-Co V-2 pseudovirus.Q-PCR was used to evaluate the neutralizing activity of Bsc Ab 16-3022 and antibody cocktail against authentic virus.The Discovery Studio2016 Client software was used to simulate the binding of three sc Fvs to SARS-COV-2RBD,and the epitopes of sc Fvs to SARS-COV-2 RBD were analyzed by competitive ELISA.Results:All five constructs were expressed as inclusion body in E.coli and target proteins at a purity over 90%were obtained after purification on Ni-NTA column.ELISA results showed that all the five antibodies had high affinity with SARS-COV-2RBD,and the binding ability of Bsc Abs was 4-8 times higher than that of sc Fvs,which was consistent with the results of Western blot.MTT results illustrated that none of the five antibodies had cytotoxicity to Vero cells in the concentration range of 0-10μM.The pseudovirus neutralizing results showed that Bsc Abs and antibody cocktail significantly improved the neutralizing ability against SARS-COV-2 pseudovirus compared with sc Fvs,in which the IC₉₀of Bsc Ab 16-3022 was lower than 43 n M,192 times better than that of S1-16 and S3022.Q-PCR results showed that 16-3022 and antibody cocktails at the concentration of 10μM can effectively inhibit the genome copy number of SARS-COV-2,in which 16-3022 can reduce the copy number of virus genome by 58times.The results of molecular docking showed that the three sc Fvs could bind to different regions of SARS-COV-2 RBD respectively,and the prediction results were verified by competitive ELISA.Conclusion:1.Using a prokaryotic expression system,we have successfully expressed and evaluated the Bsc Ab with high affinity and neutralizing activity against SARS-COV-2,which combined the advantages of low cost and high expression efficiency.2.We predicted and verified that the epitopes of the three sc Fvs bound to SARS-COV-2 RBD did not completely coincide,and S3022 could play a synergistic role with other SARS-COV-2 RBD targeting antibodies to enhance neutralizing activity,which provides a new idea for the subsequent antibody development against SARS-COV-2.