Advancing cancer immunotherapy by exploring NK cell education, antibody targeting and tissue homing : stay tuned for directions reaching beyond the natural biology

Cancer immunotherapy is a fast-growing field. The natural killer (NK) cells’ innate ability to detect malignant cells and capacity to perform antibody-dependent cellular cytotoxicity (ADCC) together with low risk of inducing side effects makes these cells an attractive tool for cancer immunotherapy. NK cells can be used in both allogeneic and autologous settings and allow for adoptive infusion. To this end, NK cells can be short-term activated or ex vivo expanded before infusion. In order to achieve clinical responses, approaches to promote NK cell cytotoxicity, persistence, as well as homing to the tumor bearing tissue are likely key. The work presented in this thesis aims to explore NK cell education, antibody targeting and tissue homing in order to further advance NK cell-based cancer immunotherapy. In paper I, we comprehensively investigated how the functional hierarchies of NK cell education relates to the NK cells capacity to perform ADCC. Data shows that ADCC triggered by a monoclonal or a bispecific antibody is subordinate to NK cell down-tuning hierarchies and tolerance but not directly augmented by inhibitory KIR-education tuning up of missing-self reactivity. Additionally, we established a positive association between CD16 expression levels and KIR-mediated education status. This work also identified that NKG2Aexpressing NK cells mediate superior ADCC, despite expressing lower levels of CD16. Overall, paper I identifies novel resolution to NK cell education hierarchies relevant for both fundamental understanding of NK cell education and therapeutic applications. In paper II, we compared tumor targeting and ADCC of resting, short-term IL-2 activated and ex vivo expanded NK cells. By assessing the education status of NK cell subsets, we uncovered that LIR-1 could educate ex vivo expanded NK cells while resting LIR-1- expressing NK cells were hyporesponsive against missing-self tumor cells. In contrast to other educated NK cell subsets, LIR-1-expressing ex vivo expanded NK cells were able to break tolerance to tumor cells expressing HLA class I molecules when combined with the monoclonal antibody rituximab. Its superior ADCC capacity while being inhibited by most HLA class I molecules suggests that LIR-1-expressing NK cells mark a potentially safe and attractive subset to be further explored in NK cell-based cancer immunotherapy. In paper III and IV we look beyond the natural biology with the intention to equip ex vivo expanded NK cells with specific tissue-trafficking molecules to redirect the migration of infused NK cells towards lymphoma- or leukemia-bearing tissues. Paper III provides proofof- concept that mRNA electroporation of ex vivo expanded NK cells to overexpress the chemokine receptors CCR7 and CXCR5 and the adhesion molecule CD62L increase in vivo homing to the lymph nodes in a mouse model. In vitro data showed that lymphoma patientderived NK cells could be equally well expanded and modified for improved homing and further equipped with high affinity CD16 to improve ADCC against autologous tumor biopsies. In paper IV, we applied biological insights from a genetic condition where patients carrying a mutation in the CXCR4 chemokine receptor suffer from lymphocytes being retained in the bone marrow. Introduction of mutant CXCR4-R334X mRNA to ex vivo expanded NK cells resulted in increased in vivo homing to the bone marrow in a non-tumor bearing mouse model. In summary, the first two papers identify novel NK cell education hierarchies that can be applied to harness the NK cell cytotoxicity against cancer alone or in combination with a tumor targeting antibody. In the latter papers we present translational approaches to enhance tissue-homing of adoptively infused NK cells. In conclusion, this thesis provides novel insights into NK cell biology with the potential to improve NK cell-based cancer immunotherapy