Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

Fig. 1.. Plasmablasts produce potent antibodies against SARS-CoV-2 with diverse V genes and limited mutations. A, VH, VK and VL gene usage of antibodies from plasmablasts and memory B cells (MBCs). Up to the top four genes in each chart are shown with different colors (genes that were tied for 4th and lower are not highlighted). B, Tukey’s plots showing heavy and light chain gene mutations of antibodies from plasmablasts and MBCs. Percent mutations were compared with the Mann-Whitney U-test. The middle line shows the median, and the box extends from the 1st to 3rd quartile. C, Top 21 neutralizing antibodies (IC50 <1 μg/mL) by antigen specificity (left), and neutralization curves of selected antibodies (right). D, SARS-CoV-2 neutralization potency versus heavy chain mutation levels of antibody panel. The 5 most potent antibodies are shown as solid circles. E, Neutralization potency of antibodies by cell type. Top values indicate percentages of non-neutralizing antibodies. Horizontal bars indicate mean values; Mann-Whitney U-test (non-neutralizing antibodies excluded from calculation). The 5 most potent antibodies are shown in color. F, SARS-CoV-2 neutralization curves of benchmark…

Fig. 2.. Potent antibodies against SARS-CoV-2 target diverse epitopes on the RBD and NTD and neutralize emerging variants. A, Isoaffinity plot of antibodies targeting SARS-CoV-2 RBD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. B, Neutralization potency versus affinity of anti-RBD antibodies. C, Isoaffinity plot of antibodies targeting SARS-CoV-2 NTD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. D, Neutralization potency versus affinity of anti-NTD antibodies. E, Epitope binning of anti-RBD antibodies (representative of n = 2 experiments). ACE2 was only used as an analyte (competitor) and not as a ligand, while all other antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one-way competition. The number and percentage of neutralizing antibodies (IC50 < 10 μg/mL) in each bin are…

Fig. 3.. Crystal structure of SARS-CoV-2 RBD in complex with CV503. A, CV503 binds to the ridge region of SARS-CoV-2 RBD. The heavy and light chains of CV503 are shown in orange and yellow, respectively. SARS-CoV-2 RBD is in white, where its ridge region (residues 471–491) is shown in blue. B, The ACE2/RBD complex structure (PDB ID: 6M0J)52 is superimposed on the CV503/RBD complex. The heavy chain of CV503 (orange) would clash with ACE2 (green) if bound to RBD simultaneously (indicated by red circle). C-D, Epitope of CV503. Epitope residues contacting the heavy chain are in dark blue and light chain are in light blue, while residues contacting both heavy and light chains are in ocean blue. In C, CDR loops that are directly involved in RBD-binding are labeled. In D, epitope residues are labeled. Epitope residues that are also involved in ACE2 binding are labeled in red. E, ACE2-binding site on the RBD are in light pink. ACE2 is represented as semi-transparent cartoon in pale green. Epitope residues and ACE2-interacting residues are defined as those with a buried surface area (BSA) > 0 Å2. F, F486 at the ridge region of SARS-CoV-2 RBD (blue) is clamped in a hydrophobic pocket formed by the…

Fig. 4.. Bispecific antibodies utilize both binding sites to potently neutralize SARS-CoV-2 and are effective against emerging variants. A, Scheme of DVD-Ig™. In our bispecific antibody naming system, the first name refers to the antibody used to make the outer binding site and the second refers to the antibody at the inner binding site. GS or EL refers to the type of linker connecting the two antigen-binding sites. See Materials and Methods for details. B, Binding of individual and bispecific antibodies to various domains from SARS-CoV-1 and SARS-CoV-2 (representative of n = 2 experiments). Area under the curve (AUC) values are shown after subtraction with the negative control antigen. C, Neutralization potency of bispecific antibodies with SARS-CoV-2 authentic and pseudotyped virus (MLV). Values are averaged from two experiments done in duplicate. D, Neutralization curves of CV1206_521_GS with SARS-CoV-2 authentic and pseudotyped virus. Curves are from a representative experiment, IC50 values for authentic virus are the average from two experiments and those for the pseudovirus are from an average of two (bispecific) or three (regular antibody) experiments. E, Neutralization potency of…

Fig. 5.. Bispecific antibodies prevent disease mediated by WT or E484K SARS-CoV-2 in an in vivo model. A, Weight change in hamsters that were administered CV1206_521_GS IP at a dose of 2.5 or 10 mg/kg, 24 h prior to IN virus exposure at 5log10 PFU. Differences between groups that were given the antibody versus PBS were determined using a mixed-effects repeated measures analysis with Dunnett’s multiple comparisons; **P<0.01, ***P<0.001, ****P<0.0001. n = 12 hamsters per group for days 0–3, n = 6 per group for days 4–7. Points represent mean +/− standard deviation (SD). B, Blinded clinical score assigned to hamsters throughout the course of disease. A, AM; P, PM. C, Weight change in hamsters that were administered bispecific antibodies at 1 mg/hamster, 12 h prior to IN virus exposure at 5log10 PFU. Differences between groups that were given the antibody versus PBS were determined using a mixed-effects repeated measures analysis with Dunnett’s multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n = 5 hamsters per group. Points represent mean +/− standard deviation (SD).