15353-j-veluchamy

4 NK cell ADCC enhances treatment efficacy in colorectal cancer | 105 RAS wt ), SW480 cells (EGFR + , RAS mut ) and COLO320 cells (EGFR - , RAS wt ) demonstrated that cetuximab alone induced cytotoxicity only in EGFR + RAS wt tumor cells (Figure 2A and supplementary figure 1C and D). In a next set of experiments, NK cells were combined with cetuximab and this was shown to enhance the lysis of EGFR +++ RAS wt tumor cells. Using NK-FcR blocking assays this enhancement was shown to be mediated through ADCC (Figure 2D and supplementary figure 2A-C). Investigating the role of NK cell CD16a (FcγRIIIa) polymorphisms in relation to cetuximab efficacy, we did not see a statistically significant difference between V/F and V/V donors with respect to the induction of NK cell degranulation or tumor target cell lysis. These data are in line with the recent clinical study observations published by Mellor et al, concluding that FcγRIIIa genotype differences were not predicting significant therapeutic benefits for cetuximab 26 . Of interest, our data demonstrated that NK cells were capable of also killing EGFR - RAS wt cells, EGFR + RAS wt , EGFR + RAS mut and EGFR + BRAF mut cells. Furthermore, though cetuximab was completely ineffective as monotherapy against SW480 (EGFR + RAS mut ), SW620 (EGFR + RAS mut ) andHT-29 (EGFR + RAS wt BRAF mut ) cells, cetuximab could increase the target cell killing of NK cells in this setting. The ability of NK cells to lyse tumor targets independent of EGFR, RAS and BRAF status combined with the observation that cetuximab can enhance this cytotoxic effect in EGFR expressing tumors regardless of RAS and BRAF mutational status is an added advantage as it can result in effective target cell lysis of tumors responsive or non-responsive to cetuximab monotherapy. In the clinics, the eligibility criteria for anti- EGFR therapy is based on RAS wt status, whereas the CRC patients are not evaluated for EGFR expression levels; the fact that NK cells can effectively target EGFR - tumor cells could provide an ideal platform to treat metastatic CRC patients having variable levels of EGFR expression. We observed that although NK cells can efficiently kill EGFR +/- RAS wt / mut cells (A431, COLO320, Caco-2 and SW620), its efficacy was relatively low on EGFR + RAS mut SW480 and EGFR + RAS wt BRAF mut HT-29 cells. This difference could be related to the differential expression of the inhibitory non-classical HLA-E; i.e. RAS mut SW480 and BRAF mut HT-29 cells were found to have high expression of HLA-E, an inhibitory ligand for the NK cell inhibitory receptor NKG2A (supplementary figure 3), making them less susceptible to NK cell killing 27-29 . Of interest, though NK cell induced tumor cell lysis was still lowest in SW480 and HT-29, cetuximab appeared to at least in part bypass the inhibitory effect mediated through NKG2A/HLA-E interactions, probably by tipping the balance of activating and inhibitory signals more towards NK cell activation through binding of cetuximab to the activating FcγRIIIa. Furthermore, blocking HLA-E on SW480 and HT-29 cells could pave the way for more effective NK cell killing and could hence translate into superior cell death when combined with cetuximab in this setting 30,31 .