15353-j-veluchamy

76 | Chapter 3 associated NK cell dysfunctionality and the inherent resistance to cytolysis of cancer cells. Clinical studies exploring the use of ex vivo expanded allogeneic PBNK from healthy donors also yielded low antitumor efficacy 28,29 , which may have been due to their limitations in terms of cell yield, purity, ability to expand in vivo , and cytotoxic capacity 30. An attractive alternative approach would be the use of umbilical cord blood CD34 + stem cell derived NK cells (UCB-NK), which are feeder cell-free cultures that can be differentiated and efficiently expanded up to 10.000 fold, resulting in a highly pure product with a high cytolytic capacity 31 . Yet another alternative might be to enhance PBNK cell-mediated cytolysis of cervical tumor cells by the tumor-targeted IgG1 monoclonal antibody CET, to invoke antibody dependent cell mediated cytotoxicity (ADCC) 32 . In this comparative study we explored the anti-tumor efficacy of two clinically applicable therapeutic strategies, i.e. UCB-NK vs. allogeneic PBNK+CET, for cervical cancer. Of note, the combination with CET is not a viable option for UCB-NK as in vitro they do not express sufficient levels of the required Fc receptor CD16 to obtain functional benefit 33-34 . A series of in vitro NK cytotoxicity assays was conducted to compare anti-tumor potency of PBNK from healthy volunteers, with or without co-incubation with CET with that of umbilical cord blood derived NK cell (UCB-NK) monotherapy against various cervical cancer cell lines. These cell lines (n=10) were stratified based on infection with different HPV types, histological origins, and differential expression levels of NK activating- and inhibitory ligands. The findings from this pre-clinical study strongly support the use of allogeneic UCB-NK derived from umbilical cord precursor cells and outline the advantages of their use as monotherapy in the treatment of cervical cancer. Results Comparative analysis of NK cell cytotoxic activity against cervical cancer cell lines Initially, we compared the anti-tumor potency of healthy activated PBNK in the presence or absence of CET. Ten cervical cancer cell lines (EGFR-expressing except for C33A, and all RAS wt ; Table 1) were subjected to PBNK only, CET only, or to a combination of PBNK with CET in order to examine ADCC effects. In line with previous studies, CET as monotherapy did not induce cell death in any of the cell lines tested (data not shown). However, cervical cancer cell lines were sensitive in varying degrees to PBNK-induced cell lysis (Figure 1a), independent of their EGFR expression levels (Figure 1b), with consistently and significantly higher cytotoxicity rates when coated with CET (P = 0.002) (Figure 1c). C33A (EGFR- cell line) was the only cell line that did not display a higher cytotoxicity rate when PBNK were combined with CET (Figure 1a-c).