Constant domain shuffling suggests no significant contribution by CH1 to direct cytotoxicity (PI uptake, HCT15, A; proliferation inhibition on COLO205, B). In contrast, CH3 (1m3 and 3h3) contributes significantly to direct cytotoxicity, with a minor contribution by mCH2 only evident in a loss-of-function approach (3h2) (PI uptake, HCT15, C; proliferation inhibition, COLO205, D). Significance versus the respective parental constructs was deduced from two-way ANOVA. Significantly increased avidity and decreased off-rate by 1m3; with significantly decreased avidity and increased off-rate by 3h2 and more pronounced by 3h3, confirming the major CH3 and minor CH2 contributions (E, F). Significance deduced using one-way ANOVA, with Dunnett’s corrections for multiple comparisons.