Glab‐ampai K.Kaewchim K.Saenlom T.Thepsawat W.Mahasongkram K.Sookrung N.Chaicumpa W.Chulanetra M.Mahidol University2023-06-182023-06-182022-06-01International Journal of Molecular Sciences Vol.23 No.12 (2022)16616596https://repository.li.mahidol.ac.th/handle/20.500.14594/83716Broadly effective and safe anti‐coronavirus agent is existentially needed. Major protease (3CLpro) is a highly conserved enzyme of betacoronaviruses. The enzyme plays pivotal role in the virus replication cycle. Thus, it is a good target of a broadly effective anti‐Betacoronavirus agent. In this study, human single‐chain antibodies (HuscFvs) of the SARS‐CoV‐2 3CLpro were generated using phage display technology. The 3CLpro‐bound phages were used to infect Escherichia coli host for the production the 3CLpro‐bound HuscFvs. Computerized simulation was used to guide the selection of the phage infected‐E. coli clones that produced HuscFvs with the 3CLpro inhibitory potential. HuscFvs of three phage infected‐E. coli clones were predicted to form contact interface with residues for 3CLpro catalytic activity, substrate binding, and homodimerization. These HuscFvs were linked to a cell‐penetrating peptide to make them cell‐penetrable, i.e., became superantibodies. The su-perantibodies blocked the 3CLpro activity in vitro, were not toxic to human cells, traversed across membrane of 3CLpro‐expressing cells to co‐localize with the intracellular 3CLpro and most of all, they inhibited replication of authentic SARS‐CoV‐2 Wuhan wild type and α, β, δ, and Omicron variants that were tested. The superantibodies should be investigated further towards clinical application as a safe and broadly effective anti‐Betacoronavirus agent.Biochemistry, Genetics and Molecular BiologyArticleSCOPUS10.3390/ijms231265872-s2.0-851320726451422006735743031