Abstract
Natural killer (NK) cells are an innate immune cell subset with promising immunotherapeutic potential due to their intrinsic ability to recognise and lyse tumour cells. NK cells can be engineered with chimeric antigen receptors (CAR) to enhance tumour recognition and destruction. Additionally, NK cells express CD16, a receptor that recognises the Fc regions of therapeutic monoclonal antibodies (mAb) that bind and inhibit immunosuppressive molecules on tumour cells. CD16 interactions with therapeutic mAb provoke NK cells to release cytokines and cytotoxic granules to destroy cancer cells.
Immunotherapy with NK cells is limited by their low prevalence within peripheral blood. Artificial antigen presenting cells (aAPCs) are genetically modified to express surface molecules that stimulate NK cell division and can expand NK cells for immunotherapy. aAPCs are typically generated through virus-based transduction methods requiring iterative cell sorting and cloning, additional safety procedures with associated costs, thus limiting their uptake and usefulness to researchers.
This research employed the Sleeping Beauty transposon system as a low risk, accessible option for generating aAPCs to expand NK cells. By including multiple genes per vector, the simultaneous expression of multiple surface molecules through one transfection was possible. Flow cytometry confirmed that aAPC cell lines successfully expressed high levels of the co-stimulatory domains 4-1BBL and CD86, and either membrane-bound (mb)IL-15 or both mbIL-15 and mbIL-21.
aAPCs expressing both mbIL-15 and mbIL-21 were effective at rapidly expanding NK cells from peripheral blood, giving a 450-fold increase in NK cell numbers in 14 days. To synthesise therapeutic mAb to test CD16-mediated cytotoxicity, the expression cell lines, Expi293F and ExpiCHO, were transfected with vectors encoding the heavy and light chains for Avelumab – an immune checkpoint inhibitor that targets PD-L1 on tumour cells. Avelumab was successfully expressed and purified from both cell lines and subsequently shown to be functional by flow cytometry. Luciferase cytotoxicity assays then demonstrated that the aAPC-expanded NK cells displayed high levels of cytotoxicity towards tumour cells in vitro. Furthermore, the addition of Avelumab increased this tumour-targeted killing. In a pilot experiment, expanded NK cells were successfully transduced with an anti-HER2 CAR, demonstrating the usefulness of this new expansion method for generating CAR NK cells. Overall, this study generated an aAPC cell line that was highly effective at expanding cytotoxic NK cells for CAR transduction, while also demonstrated that targeting tumour-expressed PD-L1 with Avelumab enhanced NK cell-mediated tumour killing, thus increasing the feasibility of NK cell immunotherapy in the future.