Binding of Fab CD3 –Fab HER2 –Fab PD-1 –biotin CiTE 27 and Fab HER2 –Fab CD3 –biotin BiTE 12 to T cells from one donor, after CD3 blockade, detected by flow cytometry, normalized to maximum binding. Data are represented as individual data points from three replicates (except in f and for BiTE 12 binding in i , which are single data points without replicates). Statistical analysis was carried out with a two-way analysis of variance (ANOVA) followed by a post hoc Šídák’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. For a and c , the differences were not significant and significant (****) at all concentrations, respectively. Curves are fitted with nonlinear regression with the following models: one site -- specific binding ( b , c , h ); [Inhibitor] versus response (three parameters) ( d , e ); [Agonist] versus response (three parameters) ( g , i ). List of P values for CiTE 24 versus BiTE 12 in b : 0.9985 (at 0.094 nM), >0.9999 (at 0.188 nM), 0.9860 (at 0.375 nM), >0.9999 (at 0.75 nM), 0.9316 (at 1.5 nM), >0.9999 (at 3 nM), 0.9303 (at 6 nM), 0.6189 (at 12 nM) and >0.9999 (at 24 nM). List of P values for CiTE 24 versus BiTE 12 in c : >0.9999 (at 0.018 nM), >0.9999 (at 0.055 nM), >0.9999 (at 0.165 nM), >0.9999 (at 0.494 nM), 0.9936 (at 1.48 nM), >0.2870 (at 4.44 nM), <0.0001 (at 13.3 nM), <0.0001 (at 40 nM) and <0.0001 (at 120 nM). List of P values for CiTE 24 versus BiTE 12 in d : <0.0001 (at 0.018 nM), <0.0001 (at 0.055 nM), <0.0001 (at 0.165 nM), <0.0001 (at 0.494 nM), <0.0001 (at 1.48 nM), <0.0001 (at 4.44 nM), <0.0001 (at 13.3 nM), <0.0001 (at 40 nM) and <0.0001 (at 120 nM). List of P values for T cell versus PBMC in e : 0.0044 (at 0.018 nM), <0.0001 (at 0.055 nM), <0.0001 (at 0.165 nM), <0.0001 (at 0.494 nM), <0.0001 (at 1.48 nM), <0.0001 (at 4.44 nM), 0.2744 (at 13.3 nM), 0.9392 (at 40 nM) and 0.9959 (at 120 nM). List of P values for CiTE 27 versus BiTE 12 in g : >0.9999 (at 0.023 nM), >0.9999 (at 0.047 nM), >0.9999 (at 0.094 nM), >0.9999 (at 0.188 nM), 0.9982 (at 0.375 nM), 0.7945 (at 0.75 nM), 0.1950 (at 1.5 nM), <0.0001 (at 3 nM), <0.0001 (at 6 nM), <0.0001 (at 12 nM) and <0.0001 (at 24 nM). List of P values for CiTE 27 versus BiTE 12 in h : >0.9999 (at 0 nM), >0.9999 (at 0.094 nM), 0.9991 (at 0.188 nM), 0.9998 (at 0.375 nM), 0.9987 (at 0.75 nM), 0.9756 (at 1.5 nM), 0.9834 (at 3 nM), 0.5546 (at 6 nM), 0.0116 (at 12 nM) and <0.0001 (at 24 nM). List of P values for CiTE 27 versus BiTE 12 in i : 0.9123 (at 0 nM), 0.6874 (at 0.188 nM), 0.8176 (at 0.375 nM), 0.3741 (at 0.75 nM), 0.0169 (at 1.5 nM), 0.0028 (at 3 nM), <0.0001 (at 6 nM), <0.0001 (at 12 nM) and <0.0001 (at 24 nM).
