Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: 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 4 th 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 1 st to 3 rd 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 antibodies from different groups , , . G, Neutralization IC50 values of antibodies in our panel and benchmark antibodies in three different neutralization assays. Authentic SARS-CoV-2 FRNA values are from a single experiment done in quadruplicate, authentic SARS-CoV-2 (Scripps) values are an average of two experiments done in duplicate, pseudovirus (MLV) values are an average of 3 experiments in duplicate. The colors indicate the different sources of the antibodies: red, this study; blue, ref. 21; yellow, ref. 11; green, ref. 18.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: MANN-WHITNEY, Neutralization, Mutagenesis

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: 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 shown. F , Epitope bins represented by C135 (yellow bin), S309 (purple bin), ACE2 (red bin), CR3022 (cyan bin), as well as the NTD-specific antibody 4-8 (orange) modeled onto a SARS-CoV- 2 spike protein (white cartoon). Antibody 4-8 was not binned successfully in our experiments but binds to a similar region to 2-17 and 5-24 (ref. 5), which were binned. The epitope sites are color-coded the same as in and . N-glycans at the N343 glycan site are represented by sticks. G , Epitope binning of anti-NTD antibodies (representative of n = 2 experiments). All 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 shown. H , Binding of mAb panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). I , Neutralization potency of CV503, CV664 and CV993 against B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets, and numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Neutralization, Binding Assay

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: 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) 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 Å 2 . F , F486 at the ridge region of SARS-CoV-2 RBD (blue) is clamped in a hydrophobic pocket formed by the heavy (orange) and light chains (yellow) of CV503.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Binding Assay, Labeling

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , Scheme of DVD-Ig TM . 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 CV1206_521_GS versus a cocktail of CV1206 and CV521, with concentrations shown in the molar scale to enable a fair comparison. For the antibody combination, the values on the x-axis refers to the concentration of each antibody in the cocktail, e.g. 10 nM refers to 10 nM of CV1206 + 10 nM of CV521. F , 3D reconstruction of CV1206_521_GS from nsEM images. Two “one RBD up” models (PDB 6VYB) in green are docked into the reconstruction. Similarly, multiple mock scFv’s in orange and purple were docked to approximate the DVD-Ig molecule. O, outer binding site; I, inner binding site. G , Binding of bispecific antibody panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). H , Neutralization potency of bispecific antibodies against D614G, B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets: numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type. ND, not determined. I, Potency of CV503_664_EL versus individual component mAbs against wild-type and B.1.351 SARS-CoV-2 pseudotyped virus (lentivirus).

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Binding Assay, Negative Control, Neutralization, Concentration Assay