Journal: iScience

Article Title: Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

doi: 10.1016/j.isci.2024.109363

Figure Lengend Snippet: Anti-RBD CvMab-6 and anti-S2 CvMab-62 epitopes (A) Amino acid alignment of CvMab-6 target element. Amino acid residues corresponding to position 459–478 of the Wuhan-Hu-1 spike protein are aligned with those of bat coronaviruses RaTG13 and Khosta2. (B) Western blot analysis of CvMab-6 on bat coronavirus spike proteins. CvMab-6 did not react with the Khosta2 spike protein. (C) Indirect immunofluorescence analysis of CvMab-6 on bat coronavirus spike proteins. CvMab-6 did not react with the Khosta2 spike protein expressed in HEK293 cells. (D) Structural model of the CvMab-6 target element. The red region represents the CvMab-6 binding site. (E) CvMab-6 did not inhibit RBD-ACE2 binding. Preincubation of RBD with bebtelovimab, but not with CvMab-6, inhibited in vitro binding between RBD and ACE2 using ELISA. Data are presented as the means ± SD (n = 3). (F) Amino acid alignment of the CvMab-6 target element corresponding to positions 459–478 of the several variant spike proteins. Light blue indicates mutated amino acids. (G) Western blot analysis of CvMab-62 on bat coronavirus spike proteins. The reactivity of CvMab-62 with the Khosta2 spike protein was strongly reduced compared to the anti-S2 1A9 antibody. (H) Indirect immunofluorescence analysis showed reduced reactivity of CvMab-62 on bat coronavirus Kohosta2 spike proteins expressed in HEK293T cells. (I) Summary of western blot analysis of the deletion mutant S2 proteins using CvMab-62. CvMab-62 detected a deletion mutant S2 protein lacking amino acid residues 1149–1162, but did not react with the D1146E mutant S2 protein. (J) Amino acid alignments of corresponding to positions 1070–1173 of the Wuhan-Hu-1 spike protein were aligned with those of bat coronaviruses RaTG13 and Khosta2. (K) Amino acid alignments of the CvMab-62 target element corresponding to positions 459–478 of the several variant spike proteins. Light blue indicates mutated amino acids.

Article Snippet: Finally, enzymatic reactions were performed using an ELISA POD substrate TMB kit (Cat# 05298-80, Nacalai Tesque).

Techniques: Western Blot, Immunofluorescence, Binding Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Variant Assay, Mutagenesis

Journal: iScience

Article Title: Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

doi: 10.1016/j.isci.2024.109363

Figure Lengend Snippet: Anti-SARS-CoV-2 effects of bispecific antibodies (A) Schematic of bispecific antibodies. The scFv of anti-S2 CvMab-62 was fused to the C-terminus of anti-RBD CvMab-6 heavy (Bis1) or light (Bis2) chains. Conversely, the scFv of anti-RBD CvMab-6 was fused to the C-terminus of the anti-S2 CvMab-62 heavy (Bis3) or light (Bis4) chains. (B) The presence of recombinant bispecific antibodies was confirmed by SDS-PAGE, followed by Coomassie blue staining. (C) In vitro binding of bispecific antibodies to monomeric RBD consists of amino acids 319–541, measured by ELISA. The RBD protein (WT: Wuhan type, or BA.1) was coated in the wells, and bispecific antibodies at the indicated concentrations were added. The calculated KD values using GraphPad Prism9 are presented in the table below. (D) In vitro binding of bispecific antibodies to the trimeric spike ectodomain, was measured using ELISA. The trimeric spike ectodomain proteins (WT: Wuhan type, BA.1, BA.5.2, or BA.2.75) were coated onto the wells, and bispecific antibodies at the indicated concentrations were added. The KD values, calculated using GraphPad Prism9 are presented in the table on the right. (E) Neutralization of bispecific antibodies against SARS-CoV-2 pseudotyped viruses. Pseudotyped viruses were preincubated with antibodies at the indicated concentrations and then used to infect VeroE6/TMPRSS2 cells. Three days post-infection, cellular luciferase activity was measured to estimate the pseudotyped virus infection ratio. Data are presented as the means ± SD (n = 3). The inhibitory effects of the bispecific antibodies are shown as IC 50 values summarized in the table on the right side. ND: not determined. (F) Neutralization activity of bispecific antibodies against authentic SARS-CoV-2 viruses. Authentic SARS-CoV-2 variant viruses were preincubated with antibodies at the indicated concentrations and then used to infect VeroE6/TMPRSS2 cells. At 24 h post-infection, viral genomic RNA in cells was measured by quantitative RT-PCR, and viral replication was shown as the ratio of the control. Data are presented as the means ± SD (n = 4). The inhibitory effects of the bispecific antibodies are shown as IC 50 values summarized in the table on the right side. ND: not determined.

Article Snippet: Finally, enzymatic reactions were performed using an ELISA POD substrate TMB kit (Cat# 05298-80, Nacalai Tesque).

Techniques: Recombinant, SDS Page, Staining, In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Infection, Luciferase, Activity Assay, Virus, Variant Assay, Quantitative RT-PCR, Control

Journal: iScience

Article Title: Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

doi: 10.1016/j.isci.2024.109363

Figure Lengend Snippet: Binding by CvMab-62 is critical for neutralizing activity of bispecific antibody (A) In vitro binding of CvMab-62 to the trimeric spike was inhibited by the novel S2 epitope. In vitro binding of CvMab-62 to the trimeric spike ectodomain, was measured using ELISA. CvMab-62 was preincubated with 1 μM of epitope peptide (D1146) or D1146E mutated peptide (E1146), and subsequently added to the trimeric spike ectodomain protein coated ELISA plate. Data are presented as the means ± SD (n = 3). Statistical differences were determined using a one-way analysis of variance (ANOVA), and a p value <0.05 was considered statistically significant, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. ns was not statistically significant. (B) Western blotting analysis of D1146E mutated spike protein. Spike proteins of Whuan-Hu-1 and D1146E mutants were expressed in HEK293T cells and detected using CvMab-6 or CvMab-62 antibodies. (C) Neutralization activity of Bis3 against the D1146E-mutanted spike-expressing pseudotyped virus. Wuhan wild-type or D1146E-mutated spike-expressing pseudotyped viruses were preincubated with Bis3 at the indicated concentrations and they were then used to infect VeroE6/TMPRSS2 cells. Three days post-infection, cellular luciferase activity was measured to estimate the pseudotyped virus infection ratio. Data are presented as the means ± SD (n = 3).

Article Snippet: Finally, enzymatic reactions were performed using an ELISA POD substrate TMB kit (Cat# 05298-80, Nacalai Tesque).

Techniques: Binding Assay, Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Neutralization, Expressing, Virus, Infection, Luciferase

Journal: iScience

Article Title: Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

doi: 10.1016/j.isci.2024.109363

Figure Lengend Snippet: Bispecific antibody constructed with bebtelovimab and CvMab-62 (A) Structural model of bebtelovimab CDR binding to SARS-CoV-2 RBD. The K444 residue in the RBD of the SARS-CoV-2 spike protein interacts with D185 and D187 in the heavy-chain CDR of bebtelovimab. The BQ.1.1 variant of the spike protein has a mutation at K444 (replaced by T), which is responsible for making it resistant to bebtelovimab. (B) Schematic of the bispecific antibody Bis-Beb. The scFv of anti-RBD bebtelovimab was fused with the C-terminus of the anti-S2 CvMab-62 heavy chains. (C) In vitro binding of the bispecific antibody to trimeric spike ectodomain of omicron BA.4/5 consisting of 1231 amino acids measured by ELISA. (D) In vitro binding of the bispecific antibody to the trimeric spike ectodomain of Omicron BQ.1.1, consisting of 1231 amino acids, as measured by ELISA. The wells were coated with the trimeric spike ectodomain protein, and bispecific antibodies at the indicated concentrations were added to evaluate antibody binding to the trimeric spike protein. (E) Summary table of the in vitro binding ability of the bispecific antibody in ELISA. The KD values were estimated using C and D by GraphPad Prism9. (F) SPR analysis of the bispecific antibody against the trimeric spike ectodomain. The spike ectodomain, either BQ.1.1 or BA. 4/5 was captured as a ligand, and two buffer conditions, pH 7.4 and pH 5.5, were tested. Antibody as an analyte, Bis-Beb (red lines), bebtelovimab (blue lines), and CvMab-62 (black lines) were tested. The response curves are representative of the two experiments. ∗The reported kinetic constant kd was outside the limits measured using the instrument in this study.

Article Snippet: Finally, enzymatic reactions were performed using an ELISA POD substrate TMB kit (Cat# 05298-80, Nacalai Tesque).

Techniques: Construct, Binding Assay, Residue, Variant Assay, Mutagenesis, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

doi: 10.1016/j.isci.2024.109363

Figure Lengend Snippet: Neutralizing ability of Bis-Beb against bebtelovimab-resistant BQ.1.1 (A) In vitro binding of the bispecific antibody to the monomeric RBD of BQ.1, was measured using ELISA. The wells were coated with the monomeric BQ.1 RBD protein, and antibodies at the indicated concentrations were added to evaluate antibody binding. The KD values estimated by GraphPad Prism9 are summarized in the table below. (B) Relative resistance to in vitro binding of Bis-Beb to the trimeric spike. In vitro binding of CvMab-62 or Bis-Beb to the trimeric spike ectodomain, was measured using ELISA. Antibodies were preincubated with the epitope peptide (D1146) or D1146E mutated peptide (E1146) at the indicated concentrations, and subsequently added to a trimeric spike ectodomain protein-coated ELISA plate. (C) Inhibition of in vitro ACE2-spike binding by the bispecific antibody was confirmed using ELISA. His-tagged trimeric spike ectodomain of BQ.1.1 was preincubated with bispecific antibodies (10 μg/mL), and then premixtures added to a well coated with recombinant ACE2 protein. After washing, the ACE2-bound trimeric spike protein was probed with anti-His-tag antibodies. Data are presented as the means ± SD (n = 3). (D) Neutralization of bispecific antibodies against SARS-CoV-2 pseudotyped viruses. Pseudotyped viruses (expressing BA.5.2- or K444T mutated BA.5.2-type spike) were preincubated with antibodies at the indicated concentrations and then used to infect VeroE6/TMPRSS2 cells. Three days post-infection, cellular luciferase activity was measured to estimate the pseudotyped virus infection ratio. Data are presented as the means ± SD (n = 3). The inhibitory effects of bispecific antibodies are shown as IC 50 values and summarized in the table below. (E) Neutralization of bispecific antibodies against authentic SARS-CoV-2. Authentic SARS-CoV-2 variants (BA.5.2.1 or BQ.1.1) were preincubated with antibodies at the indicated concentrations and then used to infect VeroE6/TMPRSS2 cells. At 24 h post-infection, viral genomic RNA in the cells was measured by quantitative RT-PCR, and viral replication shown as the ratio of the control. Data are presented as the means ± SD (n = 4). The inhibitory effects of bispecific antibodies are shown as IC 50 values and summarized in the table below.

Article Snippet: Finally, enzymatic reactions were performed using an ELISA POD substrate TMB kit (Cat# 05298-80, Nacalai Tesque).

Techniques: In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Recombinant, Neutralization, Expressing, Infection, Luciferase, Activity Assay, Virus, Quantitative RT-PCR, Control

Journal: iScience

Article Title: Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

doi: 10.1016/j.isci.2024.109363

Figure Lengend Snippet: Neutralizing ability of humanized Bis-Beb against bebtelovimab-resistant BQ.1.1 (A) In vitro bindings of the mouse-derived and humanized antibodies to the trimeric spike ectodomain of the Wuhan type was compared using ELISA. The calculated KD values using GraphPad Prism9 are presented in the table below. (B) In vitro binding of the mouse-derived and humanized antibodies to monomeric RBD or trimeric spike ectodomain proteins was measured by ELISA. The BQ.1 monomeric RBD or trimeric ectodomain proteins were coated in the wells, and antibodies at the indicated concentrations were added. The calculated KD values using GraphPad Prism9 are presented in the table below. (C) Neutralization of humanized bispecific antibodies against SARS-CoV-2 pseudotyped viruses. Pseudotyped viruses (expressing BA.5.2- or K444T mutated BA.5.2-type spike) were preincubated with antibodies at the indicated concentrations and they were then used to infect VeroE6/TMPRSS2 cells. Three days post-infection, cellular luciferase activity was measured to estimate the pseudotyped virus infection ratio. Data are presented as the means ± SD (n = 3). The inhibitory effects of the bispecific antibodies are shown as IC 50 values and are summarized in the table below. (D) Neutralization of humanized bispecific antibodies against SARS-CoV-2. Authentic SARS-CoV-2 variants (BA.5.2.1 or BQ.1.1) were preincubated with antibodies at the indicated concentrations and used to infect VeroE6/TMPRSS2 cells. At 24 h post-infection, viral RNA in the medium supernatant was measured by quantitative RT-PCR, and viral replication was calculated as the ratio of the control. Data are presented as the means ± SD (n = 4). The inhibitory effects of the bispecific antibodies are shown as IC 50 values and are summarized in the table below. The ND was not determined. (E) Mechanism of action of the bispecific antibody Bis-Beb. Bis-Beb binds to the RBD and S2 domains of the spike protein. Bis-Beb can restore the ability to inhibit binding between BQ.1.1 RBD and ACE2 and has the capacity to interfere with the subsequent membrane fusion process involving the postfusion form of the S2 component.

Article Snippet: Finally, enzymatic reactions were performed using an ELISA POD substrate TMB kit (Cat# 05298-80, Nacalai Tesque).

Techniques: In Vitro, Derivative Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Neutralization, Expressing, Infection, Luciferase, Activity Assay, Virus, Quantitative RT-PCR, Control, Membrane

Journal: iScience

Article Title: Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

doi: 10.1016/j.isci.2024.109363

Figure Lengend Snippet:

Article Snippet: Finally, enzymatic reactions were performed using an ELISA POD substrate TMB kit (Cat# 05298-80, Nacalai Tesque).

Techniques: Virus, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Bioassay, Variant Assay, Software