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130 | Chapter 5 Previous studies have pointed out that the proteasome inhibitor (bortezomib) 43 and the immunomodulatory drug (lenalidomide) 44 sensitize tumor cells to NK mediated killing. In addition, UCB-NK cell application together with bispecific or trispecific antibodies that bind to tumor and UCB-NK cell activating receptors can also increase NK cell tumor specificity 45 . Though we did not specifically assess ADCC induced by other mAbs, it is very likely that the failure of UCB-NK to mediate ADCC is a more general phenomenon as this depends on binding to CD16/FcγRIII, which was found to be expressed at only low levels in the UCB-NK cell product. However, recent data from a clinical phase 1 study with the same UCB-NK cell product in patients with AML revealed significant upregulation of CD16 on UCB-NK cells post transfusion suggesting that the UCB-NK cell product may acquire the capacity to mediate ADCC in patients following adoptive transfer 46 . Further, this phenomenon may also provide a strong rationale for combining UCB-NK cells with bispecific or trispecific killer cell engagers 47 . Taken together, these approaches can substantially increase UCB-NK cell responses to advanced solid tumors, including mCRC. In conclusion, in this study we have demonstrated the in vitro efficacy of UCB-NK cells against multiple colorectal cancer cell lines independent of EGFR expression and EGFR downstream signaling mutations, and in addition have demonstrated the in vivo antitumor efficacy of adoptively transferred UCB-NK cells against EGFR + RAS mut tumors. As the adoptive transfer of UCB-NK cells (oNKord ® ) has been shown to be safe in patients with AML (CCMO nr NL31699 & Dutch trial register no 2818), our data provide a rationale for the clinical exploration of UCB-NK cells in the treatment of mCRC. Materials and Methods Cell lines Cell lines A431 (epidermoid carcinoma), COLO320, SW480 and HT-29 (colon carcinoma) were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco's modified medium (DMEM; Invitrogen, Carlsbad CA, USA) containing 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum (FCS; Integro, Zaandam, The Netherlands). Cell cultures were passaged every 5 days and maintained in a 37°C, 95% humidity, 5% CO2 incubator. PBNK isolation and activation Peripheral blood mononuclear cells (PBMC) were isolated from the heparinized blood of healthy donors (6 males, 4 females, age range = 56-64 and CRC patients (8 males, 2 females, age range = 66-74) after written informed consent and according to protocols approved by the institutional review board of VU University Medical Center, Amsterdam (NCT01792934). Blood samples were collected at baseline and after the first cycle of first-