Human monoclonal antibodies are safe, preventive and therapeutic tools, that can be rapidly developed to help restore the massive health and economic disruption caused by the coronavirus disease 2019 (COVID-19) pandemic. By single cell sorting 4,277 SARS-CoV-2 spike protein specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody dependent enhancement and prolong half-life, neutralized the authentic wild type virus and emerging variants containing D614G, E484K and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc-functions.

SUMMARYHuman monoclonal antibodies are safe, preventive and therapeutic tools, that can be rapidly developed to help restore the massive health and economic disruption caused by the Covid-19 pandemic. By single cell sorting 4277 SARS-CoV-2 spike protein specific memory B cells from 14 Covid-19 survivors, 453 neutralizing antibodies were identified and 220 of them were expressed as IgG. Up to 65,9% of monoclonals neutralized the wild type virus at a concentration of >500 ng/mL, 23,6% neutralized the virus in the range of 100 - 500 ng/mL and 9,1% had a neutralization potency in the range of 10 - 100 ng/mL. Only 1,4% neutralized the authentic virus with a potency of 1-10 ng/mL. We found that the most potent neutralizing antibodies are extremely rare and recognize the RBD, followed in potency by antibodies that recognize the S1 domain, the S-protein trimeric structure and the S2 subunit. The three most potent monoclonal antibodies identified were able to neutralize the wild type and D614G mutant viruses with less than 10 ng/mL and are good candidates for the development of prophylactic and therapeutic tools against SARS-CoV-2.One Sentence SummaryExtremely potent neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for prophylactic and therapeutic interventions.

30In the absence of approved drugs or vaccines, there is a pressing need to develop tools for therapy 31 and prevention of Covid-19. Human monoclonal antibodies have very good probability of being 32 safe and effective tools for therapy and prevention of SARS-CoV-2 infection and disease. Here we 33 describe the screening of PBMCs from seven people who survived Covid-19 infection to isolate 34 human monoclonal antibodies against SARS-CoV-2. Over 1,100 memory B cells were single-cell 35 sorted using the stabilized prefusion form of the spike protein and incubated for two weeks to allow 36 natural production of antibodies. Supernatants from each cell were tested by ELISA for spike 37 protein binding, and positive antibodies were further tested for neutralization of spike binding to 38 receptor(s) on Vero E6 cells and for virus neutralization in vitro. From the 1,167 memory B specific 39 for SARS-CoV-2, we recovered 318 B lymphocytes expressing human monoclonals recognizing the 40 spike protein and 74 of these were able to inhibit the binding of the spike protein to the receptor. 41 Finally, 17 mAbs were able to neutralize the virus when assessed for neutralization in vitro. Lead 42 candidates to progress into the drug development pipeline will be selected from the panel of 43 neutralizing antibodies identified with the procedure described in this study. 45MBCs binding to the SARS-CoV-2 S-protein trimer in its prefusion conformation. A total of 1,167 113 S-protein-binding MBCs were successfully retrieved with frequencies ranging from 0,17% to 114 1,41% (Table 2). Following the sorting procedure, S-protein + MBCs were incubated over a layer of 115 3T3-CD40L feeder cells in the presence of IL-2 and IL-21 stimuli for two weeks to allow natural 116 production of immunoglobulins (10). Subsequently, MBC supernatants containing IgG or IgA were 117 tested for their ability to bind either the SARS-CoV-2 S-protein S1 + S2 subunits (Fig. 3A) or 118 trimer in its prefusion conformation (Fig. 3B) by enzyme linked immunosorbent assay (ELISA). A 119 panel of 318 mAbs specific for the SARS-CoV-2 S-protein were identified showing a broad range