Fig. 2

Functional characterization of humanized Muc16^ecto antibody. (a) Matrigel invasion assays performed with or without the addition of anti-MUC16^ecto antibodies. The antibody 18C6 was used as the positive control and decreased inhibition of all three cell lines. Humanized anti-MUC16^ecto 4H11 antibodies H1L1, H1L2, H2L1, and H2L2 were tested. All antibodies showed statistically significant inhibition of Matrigel invasion except OVCAR3 cells treated with H2L2. p < 0.005; * compared to OVCAR3 control, ** compared to OVCAR-433 control, *** compared to CAOV3 control, n.s: not significant. (b) 4 h Cr release cytotoxicity assays conducted with MUC16^ecto – directed second-generation CAR T-cells derived from H1L2 and H2L1 scFv sequences at the indicated effector to target (E:T) ratios. Both CAR T-cells showed dose-dependent cytotoxicity against OVCAR3 and SKOV3- MUC16^ecto tumor cells. (c) H1L2 4H11 CAR T-cells co-cultured with OVCAR3 cells and (d) SKOV3- MUC16^ecto tumor cells and evaluated for cytotoxicity after 72 h of coculture. (e) Cytokine analysis of H1L2 4H11 CAR T-cells co-cultured with SKOV3- MUC16^ecto or OVCAR3 tumor cells for 72 h. (f) 8–12-week-old female NSG mice were inoculated with SKOV3- MUC16^ecto tumor cells i.p. and treated with H1L2 4H11 CAR T-cells on day 14. Animals were subsequently monitored for development of ascites or signs of distress. ** p < 0.005. Animal experiments were performed with 4 animals per treatment group and performed twice (2 biologic replicates). (g) SKOV3, (h) SKOV3- MUC16^ecto(i) OVCAR3^MUC16KO, and (j) OVCAR3 tumor cells were treated with increasing concentrations of murine VK8, m4H11, or h4H11 MMAE-ADCs for 72 h. and evaluated for cytotoxicity. Data are plotted as means ± SEM from three independent measurements, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Coculture experiments were performed with 2–3 technical replicates and 3 biological replicates unless otherwise stated