Fig. 6: 441 induces apoptosis and ablates ligand-driven growth in combination with co-receptor-targeting mAbs.

a Twenty-four hours posttreatment, single mAbs did not reduce phosphorylation of HER2, but only of downstream signaling proteins in BT474, N-87, and HCC1419 cells as revealed by immunoblotting. Only 441 strongly reduced phosphorylation of HER2. Both 441 and TZB + hA21G additionally led to cleavage of PARP in the first two cell lines. In contrast, the TZB + PZB combination was not able to induce marked apoptosis. b Induction of cell death measured by dead-cell staining (PI-positive) as a percentage of total cell staining by membrane-permeable Hoechst-33342. Forty-eight hours posttreatment cells were stained (PI/Hoechst) and analyzed on a Lionheart HT-microscope. Treatment with 441 showed a significant increase of the dead-cell population over any other treatment across five different HER2-addicted cancer cell lines (n = 6, two-sided ANOVA with Dunnett’s correction for multiple comparison). c Ligand-stimulated cells pose a particular challenge for antibodies. LJM716 was shown to inhibit HER3-ligand-driven growth⁴¹. NRG-stimulated Calu-3 cells were kept in check at least equally well by 441 + LJM716, compared to the triple treatment of TZB, PZB, and LJM716 (n = 6 biological replicates, mean plus error bar 95% confidence interval, two-sided ANOVA with Tukey’s correction for multiple testing). d Combinations inhibit phosphorylation of HER3 and downstream kinases as shown in immunoblots. e Cetuximab (CXB) recognizes part of the EGF binding site on EGFR⁴⁴ and the 441-CBX combination was significantly more beneficial in the presence of the ligand than the cocktail of TZB, PZB, and CXB (n = 6 biological replicates, mean plus error bar 95% confidence interval, two-sided ANOVA with Tukey’s correction for multiple testing). f Immunoblots show only the 441-CXB combination prevents the phosphorylation of EGFR and hence ablates downstream signaling via AKT and ERK. Source data are available in the Source Data file.