CAD patient plasma samples induce C3 fragment deposition on normal hRBCs that is inhibited by TNT003. (A) Flow cytometry plots from hRBCs exposed to the specified plasma sample (sensitization) and subsequently exposed to 25% NHS (complement activation). All plasma samples contained 10 mM EDTA; “CAD + 10 mM EDTA” refers to NHS exposure in the presence of EDTA. Y-axis: forward scatter (FCS, as defined in the y-axis); x-axis: cell surface C3 fragments. FL1-A, the fluorescence intensity measured, which corresponds to the amount of cell surface C3 fragments as defined in x-axis. (B) Bar graphs of C3 fragment deposition on hRBCs exposed to 3 representative CAD patient plasma samples (patient 34 annotated as P34, and so on) or a normal plasma sample, and subsequently exposed to NHS in the presence of 10 mM EDTA, TNT003, or an IC IgG2a antibody. (C) Averaged concentration response curve for TNT003 inhibition of C3 fragment deposition for all CAD patient samples. IC₅₀ = 5.0 ± 1.0 μg/mL (33 ± 6 nM); n = 27 samples. Data were normalized across different patient samples by setting C3 fragment deposition occurring in the presence of NHS at 100%. Individual IC₅₀ values for each patient sample are provided in supplemental Table 1. (D) Histograms showing the effect of increasing TNT003 concentration on CA-mediated C3 fragment deposition. The shaded histogram depicts C3 fragment deposition in the absence of TNT003.