Cytotoxicity assay of Fab HER2 –Fab CD3 –Sia–biotin CiTE 24 and Fab CD3 –Fab HER2 –Fab PD-1 –biotin CiTE 27 . MDA-MB-231 cells, pre-incubated with IFN-γ to induce PD-L1 expression, were co-cultured with T cells from a single donor (E:T ratio of 2:1) and treated with 0.01–10 nM CiTE 24 , CiTE 27 or BiTE 30 . MDA-MB-231 viability was assessed 24 h following treatment via LDH assay. Statistical analysis was carried out with a two-way ANOVA followed by a post hoc Tukey’s multiple comparisons test with multiplicity-adjusted P values with α = 0.05. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. List of P values for CiTE 24 versus BiTE 30 in b : 0.0004 (at 0.01 nM), <0.0001 (at 0.1 nM), <0.0001 (at 1 nM) and 0.1791 (at 10 nM). List of P values for CiTE 24 versus BiTE 30 in c : <0.0001 (at 0.01 nM), <0.0001 (at 0.1 nM), 0.0333 (at 1 nM) and 0.7435 (at 10 nM). Data are represented as individual data points, from three replicates. Curves are fitted with nonlinear regression with the following model: [Agonist] versus response (three parameters). See Supplementary Information for the ANOVA table and comparisons between CiTE 27 and BiTE 30 , and CiTE 24 and CiTE 27 .
