Optimal Cryopreservation Conditions to Preserve Viability, Proliferation, and Lytic Function of expanded human NK cells for clinical trials

INTRODUCTION: Natural killer (NK) cells are a promising tool for adoptive immunotherapy in many disease settings.The availability of large quantities of functionally effective NK cells represents one of the major challenges for immunotherapeutic trials, and a major objective of our laboratory is to enhance NK cell number and function ex vivo for subsequent adoptive immunotherapy. We previously published a robust approach for ex vivo propagation of NK cells that has been validated for healthy donor (Denman, 2012) and patient peripheral blood (Liu, 2013), cord blood (ASH 2012), and embryonic/pleuripotent stem cell sources (Knorr 2013) and has the potential to generate far more NK cells than can be infused at one time. Cryopreservation of NK cells provides significant advantages over infusion of fresh products by 1) reducing timing constraints in matching GMP manufacturing to patient need, 2) enabling multiple infusions from single large-scale expansions, and 3) ensuring uniform characteristics for aliquots, all of which are essential for generating off-the-shelf products. However, NK cells are known to have poor survival and function after cryopreservation, and this was recently confirmed in clinical trials using standard clinical GMP cryopreservation media containing Plasmalyte, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). In contrast, our preclinical data with expanded NK cells cryopreserved in media, fetal bovine serum (FBS) and DMSO showed excellent function and no significant difference between fresh and frozen cells (Liu, 2013). Therefor, we hypothesized that serum may provide protective factors for NK cells during cryopreservation.

METHODS: NK cells from four healthy donors were expanded with weekly addition of irradiated K562 Clone9.mbIL21 feeder cells as previously described. After three weeks, cells were cryopreserved with freezing media of 40% Plasmalyte and 10% DMSO, with varying ratios of HSA and human AB serum (hAB) comprising the remainder. Cryopreserved NK cells were thawed and assessed for cell recovery, viability, proliferation, and function at 3, 24, 48, 72,120, 168 hours after thawing. Cytotoxicity was determined against 721.221 target cells. RESULTS: A linear relationship was observed between hAB content and recovery, viability, and cytotoxicity, plateauing at 40% hAB. Unstimulated proliferation after thawing was associated with higher hAB content, but re-stimulation with Clone9.mbIL21 was similar in all conditions.

CONCLUSION: In the present study, we aimed to establish the benefit of including serum in cryopreservation of expanded NK cell. We confirmed a significant effect in preserving viability, cytotoxicity, and proliferation, justifying its use in clinical trials.