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122 | Chapter 5 Here, we compared two feeder cell free allogeneic NK cell products, i.e. activated peripheral blood NK cells (A-PBNK) and cord blood stem cell derived NK cells (UCB-NK), alone or in combination with cetuximab for anti-tumor effects against RAS mut CRC. Results Highly dysfunctional NK cells in CRC patients Flow cytometry was used to determine the frequency, phenotype and functionality of NK cells in PBMC of healthy volunteers (n=10, age range 56-64, 6 males/4 females) and patients with metastatic CRC (n=10, age range 66-74, 8 males/2 females) before and after the first cycle of first line palliative chemotherapy consisting of oral capecitabine (1000 mg/m 2 , bid, day 1-14), i.v. oxaliplatin (130 mg/m 2 , day 1) and i.v. bevacizumab (7.5 mg/kg, day 1, in 4/10 mCRC patients). As illustrated in figure 1A, mCRC patients harbored on average a 20% lower percentage of CD3 - CD56 + NK cells in the total CD45 + lymphocyte population as compared to healthy controls (p<0.05). These lower NK rates, which are in line with a previous report in colorectal cancer 28 , further declined after the first cycle of chemotherapy (p<0.01). We next evaluated whether this quantitative NK cell defect was also accompanied by functional defects in the NK cell population. For this purpose, the ability of NK cells from healthy volunteers and mCRC patients to induce both natural cytotoxicity and mediate ADCC of the epidermoid carcinoma cell line A431 (MHC-I low , EGFR high , KRAS wt ) was assessed. For ADCC tumor target cells were coated with cetuximab before the addition of NK cells. It was evident that the cytotoxic potential of NK cells from mCRC patients, as reflected by degranulation (i.e. CD107a surface expression), was highly impaired both before chemotherapy and after the first cycle of chemotherapy. Though NK cells of mCRC patients were capable of ADCC, as evidenced by significant increases in degranulation when target cells were coated with cetuximab (p<0.05), levels were still low compared to those observed in healthy volunteers. (Figure 1B). Of note, although the NK cells of healthy volunteers and mCRC patients expressed similar levels of CD16 (Figure 1C), this did not translate into comparable levels of ADCC. NKp44 expression, known to reflect the activation status of NK cells, was similar between the HD and mCRC groups used in NK cytotoxicity experiments (Figure 1D). Furthermore, no significant differences were observed in expression levels of NK activating (NKG2D, NKG2C) and NK inhibiting (NKG2A, KIR2D) receptors between healthy controls and CRC patients (Supplementary figure 1).