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. 2021 Apr 2;13(4):483. doi: 10.3390/pharmaceutics13040483

Figure 2.

Figure 2

EV20/NMS-P945 shows similar properties in respect to the naked EV20 antibody. (A) In vitro binding affinity to the antigen of naked and NMS-P945-conjugated EV20 antibodies. ELISA was performed using as capture antigen human HER-3 extra cellular domain (ECD) and HRP-labelled goat anti-human IgG to detect bound EV20 or EV20/NMS-P945. (B) Cell binding affinity of naked and NMS-P945-conjugated EV20 was evaluated by flow cytometry using DU145 and MDA-MB-435 cells. Cells were harvested and stained with EV20 or EV20/NMS-P945 on ice, followed by staining on ice with PE-conjugate goat anti-human Fc as a secondary antibody. Ab sec: control cells stained only with PE-conjugate goat anti-human Fc. (C) Internalization ability of naked and NMS-P945-conjugated EV20 was evaluated by flow cytometry. A375M cells were exposed to increasing doses of EV20 or EV20/NMS-P945 for 6 h (left) or exposed for different times to 0.06 nM of EV20 or EV20/NMS-P945 (right), then cells were harvested and analyzed for surface HER-3 internalization. (D) Sk-mel 24 melanoma cells were serum-starved for 24 h, and incubated for 2 h in the presence or absence of 10 μg/mL of naked or conjugated EV20 mAb, before NRG-1β stimulation (10 min, 10 ng/mL). At the end of the incubation periods, cells were lysed and analyzed for pHER-3/HER-3 and pAKT/AKT protein levels by Western blotting.