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164 | Chapter 7 Summary The active role of NK cells in controlling cancer has been intensively studied. From the clinical trial summary provided in Table 2 from Chapter 1, it is evident that adoptive transfer of NK cells in a non-transplant setting for hematological malignancies is safe and has been shown to mediate a Graft versus Tumor (GvT) effect without causing Graft versus Host Disease (GvHD). It is well known that NK cell functions are tightly regulated by the balance between arrays of NK cell activating and inhibitory receptors. In several cancer types, NK cell activating receptors and are often down-regulated, thus limiting NK cell target killing. Hence it is essential to have a deeper understanding of the expression levels of relevant NK cell receptors and the functional status of NK cells in tumor conditions. This led us to the develop two eight-color NK cell flowcytometry (FACS) panels, one to study the NK cell phenotype and the other to study the NK cell function in PBMC (or single-cell tumor) samples from multiple centers as described in Chapter 2. The NK cell FACS panels were designed, optimized and tested across three different centers using three different flow cytometers with comparable configuration, thus providing a unique platform to generate comparable and reproducible data for multicenter clinical trials. Furthermore, this study also emphasized that cryopreserved NK cells are suitable for studying NK cell phenotypes and functions including NK cell mediated ADCC. In Chapter 3, to treat anti-EGFR/cetuximab and immunotherapy-resistant cervical cancer cells, allogeneic NK cell-based therapy was explored. The cytotoxic effects of UCB- NK cells and activated PBNK cells were compared either as monotherapy or in combination with cetuximab in an in-vitro set-up. A panel of ten cervical cancer cell lines with different histology and different HPV types were subjected to NK cell killing. All these cell lines expressed low to moderate levels of EGFR (except C33A, which was EGFR negative) and were wild type for the RAS gene, but failed to respond to cetuximab monotherapy. Upon performing NK cell cytotoxicity assays, it was evident that all cervical cancer cell lines were sensitive toNK-mediated killing, independent of tumor histology andHPV type. Interestingly, UCB-NK-mediated cytolysis rates were significantly higher than those achieved with PBNK alone and equalled those of PBNK + cetuximab. The superior cytotoxicity with UCB-NK cells correlated to their low expression levels of inhibitory KIRs, in keeping with the observed lack of inhibition by HLA-ABC expressed on the cervical tumor cells. These data point to the potential application of UCB-NK cells in the treatment of cervical cancer. In Chapter 4, we addressed a major hurdle in the treatment of colorectal cancer, i.e. resistance against cetuximab therapy. RAS mutations in the EGFR signalling pathway have left nearly half of the metastatic colorectal cancer (mCRC) population, ineligible for anti- EGFR treatment. In our set-up, using allogeneic and highly activated PBNK cells, we could