Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four (alpha to delta, including all 9 CoVs associated with human disease), and some HR2 antibodies cross-reacted not only with beta-, but also with alpha- and gammacoronaviruses (Fig. change, Adam23 we identified human neutralizing antibodies to highly conserved viral epitopes. DJ-V-159 Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four (alpha to delta, including all 9 CoVs associated with human disease), and some HR2 antibodies cross-reacted not only with beta-, but also with alpha- and gammacoronaviruses (Fig. 1F, figs. S2 and S3; table S2) (11, 15, 16). To evaluate the antibodies ability to neutralize SARS-CoV-2, we used a previously established SARS-CoV-2 pseudovirus assay (4). The most potent FP antibody (fp.006) displayed a half-maximal inhibitory concentration (IC50) of 737 ng/mL, while the best HR2 neutralizer (hr2.016) had an IC50 of 10 ng/mL, which was lower than previously reported antibodies to this region that were tested alongside (CV3C25 and S2P6; Fig. 1G; S4A; table S2) (16, 17). Select anti-FP and anti-HR2 antibodies blocked infection regardless of TMPRSS2-expression by target cells and, consistent with the view that they antagonize post-attachment events, they did not interfere with ACE2 binding to S in ELISA but inhibited cell fusion (Fig. 1, ?,HH and ?andI,I, fig. S4, A and B). As expected, based on the absence of aa changes at coldspot regions, some FP and HR2 antibodies were effective against pseudoviruses corresponding to SARS-CoV-2 VOC, against ancestral and Omicron SARS-CoV-2 and by protection experiments showing that mice treated with CoV-X4042, either as pre- or post-exposure prophylaxis, maintained body weight and displayed diminished pathology and infectious virus titers in the lungs (Fig. 5, ?,EE to ?toG).G). Therefore, bispecific antibodies composed of moieties that simultaneously target conserved neutralizing epitopes on SD1 and RBD are effective against SARS-CoV-2 in preclinical models. Open in a separate window Fig. 5. In vitro neutralization and mouse protection by the bispecific antibody CoV-X4042.(A) SPR assay of the sequential binding of immobilized antibodies to SD1-RBD protein followed by either sd1.040, rbd.042 or CoV-X4042. (B) SPR analysis showing that both arms of CoV-X4042 bind simultaneously to the same SD1-RBD molecule, since avidity is retained at decreasing SD1-RBD concentrations. Increasing normalized kd values indicate loss of avidity. Solid lines, IgG; dotted lines, Fab (see also fig. S8A). (C) Normalized relative luminescence values in cell lysates of 293TACE2 cells after infection with ancestral SARS-CoV-2 pseudovirus in the presence of increasing concentrations of CoV-X4042 or its parental monoclonal antibodies individually or as a cocktail. Isotype control in black. On the right: mean IC50 values and significance (P) when parental antibodies are compared to DJ-V-159 CoV-X4042 (n=4; Welchs t-test, two-tailed). (D) Graph with IC50 values of bispecific and parental monoclonal antibodies neutralizing pseudoviruses corresponding to the indicated VOC. Mean of two independent experiments. (E) In vitro neutralization of SARS-CoV-2 by CoV-X4042. (F) CoV-X4042 protects in vivo. Top, diagram of the experiments timeline. Bottom, mouse weight over time after challenge, with ancestral SARS-CoV-2, of mice treated with antibodies either 24 hours before (PRE; n=5 per group, p=0.0079), or 2 hours after (POST; n=5 per group, p=0.0079; day 7) the infection. Mann-Whitney U test, standard deviation is shown. (G) CoV-X4042 reduces viral titers in the lungs. Mice were treated with antibodies 24 hours before infection and virus titers evaluated on day 3 (n=5 per group DJ-V-159 p=0.0079 with both ancestral and Omicron BA.1;.