Human Superantibodies to 3CL pro Inhibit Replication of SARS-CoV-2 across Variants.

Broadly effective and safe anti-coronavirus agent is existentially needed. Major protease (3CL pro ) 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 3CL pro were generated using phage display technology. The 3CL pro -bound phages were used to infect Escherichia coli host for the production the 3CL pro -bound HuscFvs. Computerized simulation was used to guide the selection of the phage infected- E. coli clones that produced HuscFvs with the 3CL pro inhibitory potential. HuscFvs of three phage infected- E. coli clones were predicted to form contact interface with residues for 3CL pro 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 superantibodies blocked the 3CL pro activity in vitro, were not toxic to human cells, traversed across membrane of 3CL pro -expressing cells to co-localize with the intracellular 3CL pro 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.