Each oligonucleotide containing 1 NNK codon diversifies a specific residue of the RBD protein into all possible amino acids. RBD of SARS-CoV-2. The crystal structure of the P5-22, P14-44, and RBD ternary complex clarified the unique mechanisms that underlie the excellent mutation resistance profiles of these antibodies. Second of all, polymeric IgG enhanced antibody avidity by eliminating P5-22s only hotspot, residue F486 in the RBD, therefore potently obstructing cell access by mutant viruses. Structural and practical analyses of antibodies screened using both potency assays and the candida RBD library exposed rare, ultrapotent, mutation-resistant nAbs against SARS-CoV-2. Keywords: COVID-19 Keywords: Drug screens, Drug therapy, Pharmacology Intro Neutralizing antibodies (nAbs), which Cyantraniliprole D3 display effectiveness for prophylactic as well as therapeutic RCBTB2 use, are being developed against the spike (S) protein of SARS-CoV-2 (1C8). These nAbs have shown efficacy in decreasing the viral titer in COVID-19 individuals and avoiding them from developing severe COVID-19 (9C11). REGN-COV2, Eli Lillys cocktail (12, 13), and Vir/GSKs sotrovimab have been approved for Emergency Use Authorization from the FDA. However, SARS-CoV-2, which is an RNA disease, manages to survive and spread by mutating (14). Even though mutation rate in coronaviruses is definitely slower than that in most RNA viruses, a typical SARS-CoV-2 disease accumulates approximately 2 single-nucleotide mutations per month in its genome. The continuing transmission of SARS-CoV-2 in large populations worldwide offers led to the development of variants of concern (VOCs) with increased transmissibility and possible resistance to current nAbs (15, 16). Considering that SARS-CoV-2 has only been distributing in the population for approximately 22 weeks without vaccine-induced antibody selection pressure, long term viral escape of vaccine variants may present a serious danger to efforts at closing the pandemic. Indeed, a recent finding of a greatly mutated VOC, omicron, which has 32 mutations in the spike protein, reportedly overcomes Eli Lilly and Regenerons clinically developed cocktail and poses a serious danger to vaccine-elicited immunity. Thus, development of nAbs that can conquer existing and long term viral mutation resistance is urgently needed. This necessitates the finding of broad spectrum nAbs, such as ADG-2 and S309, that can block both SARS-CoV-1 and SARS-CoV-2 cell access, in a manner that overcomes viral mutational escape as well (17, 18). However, mutations D405E, G502E/R/V, G504A/D/R/S/V, and Y505C/N/S were shown to abrogate ADG-2 binding (18), and S309 was also affected by viral mutations in the spike proteins receptor binding website (RBD) residues P337 and E340 (19, 20). Notably, the omicron variant offers Y505H in its RBD. Cyantraniliprole D3 The presence of H505 has been shown in the RBD of a closely Cyantraniliprole D3 related disease, RaTG13. Neither ADG-1 nor ADG-2 was able to bind to RaTG13s RBD (18), suggesting a risk of these so-called broad nAbs in combatting omicron. Consequently, with the objective of overcoming current and future VOCs, we extensively screened SARS-CoV-2 nAbs from dozens of convalescent donors to identify potent antibodies insensitive to amino acid substitutions in the RBD. Our attempts led to the finding of potent nAbs capable of neutralizing authentic SARS-CoV-2 viruses from convalescent individuals. More importantly, by screening the nAbs ability to bind to a mutated-RBD candida library, we recognized several mutation-resistant antibodies. Notably, P5-22 and P15-16 have only 1 1 hotspot, which is unprecedented in reported nAbs so far. Consistent with this as well as the RBDCP5-22CP14-44 ternary crystal structure, a cocktail consisting of P5-22 plus P14-44, designated IBI314, did not lose potency against prevailing VOCs in pseudoviral neutralization and authentic-virus neutralization in vitro and showed safety at low doses in vivo. Finally, polymeric P5-22 with 12 antigen fragment binding areas (Fabs) was shown to have the greatest neutralization efficacy and prevent viral escape. Results Solitary B cell cloning of weighty and light chains from PBMCs of convalescent individuals. To isolate nAbs, we acquired 44 peripheral blood samples from convalescent individuals who had recovered from COVID-19 for approximately 2 weeks, and isolated peripheral blood mononuclear cells (PBMCs) via Ficoll gradient centrifugation. Following B cell enrichment in PBMCs, we stained individual samples having a panel of B cell markers and fluorescently labeled SARS-CoV-2 S trimer protein and the S1 subunit. FACS analysis showed that after excluding plasma cells, the S1 and S trimer double-positive human population within the B cells of healthy donors without SARS-CoV-2 exposure was approximately 0 to 0.003%. Although convalescent donors experienced considerably higher percentages of S1 and S trimer double-positive cells, Cyantraniliprole D3 ranging from 0.03% to 0.18%, there was no statistically significant difference between the S proteinCreactive B cells of individuals with different examples of sign severity (Figure 1, A and B). We isolated solitary B cells by FACS into 96 individual wells comprising cell lysis buffer and an RNase inhibitor. Cognate antibody weighty chain and light chain pairs were acquired using reverse transcription PCR (RT-PCR).