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84 | Chapter 3 transfer in vivo which is accompanied by an increase in CD16a expression 52 , and this feature could be exploited to enhance tumor killing even more via ADCC using CET and other IgG 1 therapeutic antibodies. To facilitate clinical application, a GMP based NK cell expansion and differentiation protocol has already been established, approved by regulatory authorities and applied in a Phase-I clinical trial for elderly Acute Myeloid Leukemia patients and numbers of over 30 . 10 6 /kg body weight cytotoxic UCB-NK (oNKord ® ) can easily be achieved for therapeutic purposes (CCMO no NL31699 & Dutch trial register no 2818). Therefore, it is now entirely feasible to develop clinical protocols to explore, for the first time, adoptive transfer of UCB-NK in patients with solid tumors like cervical cancer. In conclusion, our data provide a clear rationale for the use of UCB-NK to treat cervical tumors and also the possibility of using PBNK in combination with CET for EGFR-expressing tumors, with both significantly higher cytotoxicity and degranulation levels than in PBNK only conditions. Notably, treatment with UCB-NK might serve as a generally applicable treatment for cervical cancer enabled by HLA-, histology- and HPV-independent killing mechanisms. Material & Methods Cell lines Cervical cancer cell lines CSCC7, CC8, CC10A, CC10B, CC11A, and CC11B were generated in the department of Pathology of Leiden University Medical Center (The Netherlands) from primary tumors as described previously 35 . These patient-derived cell lines as well as commercially obtained cervical cancer-derived cell lines, HeLa, SiHa, CaSki and C33A (American Type Culture Collection) were maintained in DMEM (Lonza) medium containing 4.5 g/L glucose, 10% FCS (Hyclone), 10 µg/mL gentamicin and 0.25 µg/ml amphotericin B (Gibco), 100 Units Penicillin/100 Units Streptomycin/ 0.3 mg/mL Glutamine (Thermo Fisher Scientific). Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . See Table 1 for cell line characteristics. Phenotyping of cervical cancer cell lines To phenotype cervical cancer cell lines, cell suspensions in PBS supplemented with 0.1% BSA and 0.02% NaN 3 (FACS buffer) were stained for 30 min at 4°C using antibodies to HLA-ABC (clone w6/32, Immunotools) (labeled with FITC), HLA-E (clone 3D12HLA-E, eBioscience), HLA-G (clone 87G, Biolegend), EGFR (clone EGFR.1, BD Biosciences), PVR (clone SK11.4, Biolegend), MICA/B (clone 6D4, Biolegend), ULBP2/5/6 (clone #165903, R&D systems), ULBP1 (clone #170818, R&D systems) and ULBP3 (clone #166510, R&D systems) (all labeled with PE). IgG 1 , IgG 2a , and IgG 2b isotype antibodies were used as negative controls. After incubation, the cells were washed with FACS buffer and analyzed using a