Effects Of End-linked Polypeptides On The Function Of SARS-CoV-2 Neutralizing Nanobodies

The coronavirus pneumonia outbreak(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)has caused an unprecedented shock to the global economy and health.Due to the high mutation rate of RNA viruses,new challenges are presented for antibody drug development.Considering the respiratory tract infection caused by the new coronavirus first,as well as the production cost,drug bioavailability,administration method,etc.,the nanobody VHH has the advantages of small size,fast development speed,inhalation administration,and easy construction of the multivalent body to prevent immune escape,is one of the hot topics in the development of new antibodies.However,as protein drugs,nanobodies inevitably face potential problems such as stability and half-life.In this study,we designed two peptide sequences for conjugating to the ends of two nanobodies VHH72 and Nb21 targeting the viral Spike protein,to explore their effects on the function of nanobodies.One peptide is designed as the unique host protease hydrolysis site sequence PS on the S protein of SARS-Cov-2,to competitively inhibit S proteolysis and block the virus entry.The other peptide is a nonsense control NC generated by randomly rearranging the PS sequence using a software.Then,four antibody-peptide proteins,namely VHH72-PS,VHH72-NC,Nb21-PS,and Nb21-NC,were expressed and purified.In order to test the neutralizing ability,the HEK293 T cell line over-expressing ACE2 was constructed by lentivirus as the target cell,and the lenti pseudovirus with the spike protein was packaged.1.The addition of PS or NC after Nb21,a highly potent neutralizing antibody,did not significantly change its neutralizing ability,and the EC50 of Nb21-NC was slightly smaller than that of Nb21-PS.2.Adding PS or NC to the end of VHH72,a nanobody without neutralization ability can incur neutralization ability.The EC50 of VHH72-NC is smaller than that of VHH72-PS,indicating that the improvement effect of the NC sequence is more prominent.