15353-j-veluchamy

100 | Chapter 4 RAS mut ; KRAS exon 2 c.35>T; p.G12V) and HT-29 (EGFR + , RAS wt , BRAF mut ). Binding ability towards biotinylated cetuximab was negative for COLO320 EGFR (ΔMFI=1), with a relatively low EGFR expression detected on SW480 cells (ΔMFI=17) as shown in supplementary figure 1A and B. The EGFR expression level (ΔMFI) on the other colon cancer cell lines was 23 for CaCo-2, 7 for HT-29, and 3 for SW620 (data not shown). In addition, cetuximab as a single agent could not induce cytotoxicity, even at increasing concentrations of up to 1000μg/ ml (Supplementary figure 1C and D). In the next step, to test NK killing effects alone and in combination with cetuximab, all five colon cancer cell lines were coated with 5µg/ml Figure 2: Anti-EGFR mAb cytotoxicity in combination with NK cells The effect of IL-2 and IL-15 activated PBNK cells, cetuximab and panitumumab on lysis of A431 tumor cells was assessed. The percentage of ADCC was calculated based on percentage of PBSE labelled A431 cells staining positive for 7AAD in different co-culture conditions (A). Effector cells from the assay were stained with CD107a to measure NK cell degranulation by flow cytometry (B). Cell free culture supernatants were collected at the end of a 4hr co-culture and analyzed for IFNγ release by ELISA (C). The contribution of FcR mediated effector functions of NK cells (i.e. ADCC) on NK cell mediated A431 tumor cell lysis was tested by blocking the FcR receptor on NK cells (D). Data presented is from six individual PBNK donors. Columns are mean of triplicate values; with bars showing SD. Mean ± SD for each significant condition are represented as p = <0.05 *, <0.01 **, <0.005 ***, <0.001 ****.