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98 | Chapter 4 Results More potent NK effector cell activation and ADCC effected by cetuximab than by panitumumab To establish which of the anti-EGFR mAbs, cetuximab or panitumumab, exerted higher functionality with respect to EGFR recognition and cytotoxicity, both were tested on strongly EGFR positive (EGFR +++ ) A431 cells. Flowcytometric detection of EGFR using biotinylated cetuximab ( Δ MFI=217) was nearly two-fold intense than observed with biotinylated panitumumab ( Δ MFI=123), as shown in Figure1A and B. Next, A431 cells were treated with cetuximab or panitumumab to calculate the concentration required to induce 50% of maximal cytotoxicity. At higher concentrations than 1000µg/ml both mAbs were equally cytotoxic (61 ± 2%). Titrating down, the concentrations required to induce 50% of maximal cytotoxicity (EC 50 value) of EGFR +++ targets were found to be 5µg/ml for cetuximab (31 ± 2%) and 100µg/ml for panitumumab (36 ± 1%) respectively (Figure 1C). Based on these findings concentrations of 5µg/ml of cetuximab and 100µg/ml of panitumumab were used in all subsequent experiments to assess their ADCC efficacy when combined with NK cells. To this end A431 cells were coated with either cetuximab or panitumumab and co-incubated with activated NK cells. A significant increase in A431 cell death was observed over NK cells only, when NK cells were combined with cetuximab, but not with panitumumab coated targets (Figure 2A). This is consistent with the IgG 2 isotype of panitumumab, which precludes high- affinity binding to NK CD16a. In line with these ADCC data, degranulation of NK cells, as assessed by CD107a expression, was significantly increased when tumor target cells were coated with cetuximab (Figure 2B and supplementary figure 2B). Similarly, to the observed ADCC and CD107a levels, IFNγ production was increased in NK cell and cetuximab co- cultures (Figure 2C). To formally demonstrate that the cetuximab-related increase in target cell killing was due to ADCC, NK-FcR receptors were blocked using a FcR blocking reagent (Miltenyi Biotec) and then incubated with cetuximab coated target cells. As shown in Figure 2D and supplementary figure 2C, FcR blocking resulted in a considerable and statistically significant reduction in degranulation of the CD16a + NK cell compartment. Together, these data provide a clear rationale to combine NK cells and cetuximab to increase the killing of EGFR +++ targets by ADCC. NK CD16a (FcγRIIIa) polymorphism does not significantly influence cetuximab induced ADCC ex vivo Several clinical studies reported that NK FcγRIIIa receptor polymorphisms affected the clinical efficacy of cetuximab, due to a variable binding affinity to NK cells, thereby directly affecting the potency of ADCC 15,16 . In order to test if V158V (V/V) and V158F (V/F) polymorphic versions of CD16 translated to differences in ADCC upon engagement with