Figure 2
Bispecific antibodies targeting IsdB and C1q can increase complement deposition on the surface of S. aureus leading to cell death. (a) Proposed mechanism of action of the anti-IsdB x anti-C1q antibody. One arm of the antibody (orange and gray) targets the S. aureus surface antigen IsdB with high affinity. The other arm (blue and gray) targets the complement C1q protein. Engagement of multiple C1q heads by multiple bsAbs can lead to activation of complement. (b) C1q deposition on S. aureus was measured using a plate-based format. S. aureus Newman was coated onto plates and then incubated with NHS in the presence of anti-IsdB x anti-C1q bsAb, an anti-IsdB bivalent antibody or an isotype control antibody. C1q deposition was measured with a primary antibody specific for C1q and an HRP-conjugated secondary antibody. Results are plotted as mean ± standard deviation. (c) Whole blood assays were performed by incubating S. aureus Newman with blood alone (far left red column), blood with an anti-IsdB bivalent antibody (columns 2–7; purple) or blood with anti-IsdB x anti-C1q bsAb (columns 8–13; orange). The antibodies were combined with either a C5 blocking Fab (columns 3 and 9), an isotype control Fab (columns 4 and 10), or cytochalasin D (columns 5 and 11), both the C5-blocking antibody and cytochalasin D (columns 6 and 12) or both the isotype control Fab and cytochalasin D (columns 7 and 13). Bacterial growth was enumerated by plating. ****P < 0.0001, one-way ANOVA with Dunnett’s test showing samples significantly different from no treatment sample. Results are plotted as mean with standard deviation. (d) bsAbs recognizing C1q and different antigens on the surface of S. aureus were produced. Blood assays were performed as described above with two concentrations of antibody. Full length-antibodies were tested at 10 and 100 μg/ml, antibodies with an scFv arm were tested at 8.5 and 85 ug/ml (the molar equivalent of the full-length antibodies). Results are plotted as mean with standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA showing bsAb samples significantly different from the corresponding bivalent antibody.
