Cancer immunotherapy is a highly successful and rapidly evolving treatment modality that works by augmenting the body’s own immune system. While various immune stimulation strategies such as PD-1/PD-L1 or CTLA-4 checkpoint blockade result in robust responses, even in patients with advanced cancers, the overall response rate is low. While immune checkpoint inhibitors are known to enhance cytotoxic T cells’ antitumor response, current evidence suggests that immune responses independent of cytotoxic T cells, such as Natural Killer (NK) cells, play crucial role in the efficacy of immunotherapeutic interventions. NK cells hold a distinct role in potentiating the innate immune response and activating the adaptive immune system. This review highlights the importance of the early actions of the NK cell response and the pivotal role NK cells hold in priming the immune system and setting the stage for successful response to cancer immunotherapy. Yet, in many patients the NK cell compartment is compromised thus lowering the chances of successful outcomes of many immunotherapies. An overview of mechanisms that can drive NK cell dysfunction and hinder immunotherapy success is provided. Rather than relying on the likely dysfunctional endogenous NK cells to work with immunotherapies, adoptive allogeneic NK cell therapies provide a viable solution to increase response to immunotherapies. This review highlights the advances made in development of NK cell therapeutics for clinical application with evidence supporting their combinatorial application with other immune-oncology approaches to improve outcomes of immunotherapies.

132 Background: NK cells can kill malignant cells to provide innate immunity against tumors. Due to their low abundance in blood, a focus is to expand NK cells ex vivo having enhanced anti-tumor cytotoxicity to be used as a treatment. Our group has pioneered a cell-free method using plasma membrane (PM) particles derived from K562 cells expressing 41BBL and membrane-bound IL-21 (K562.mb21) which were developed for NK cell expansion. Compared to feeder cell based methods for NK cell expansion, PM21-particles improve safety and allow for potential wide-spread dissemination, and also allows direct in vivo use. Exosomes, vesicles naturally secreted by cells, may yet be another novel feeder cell free way for NK cell expansion and may have further advantageous therapeutic dimensions. Methods: EX21-exosomes and PM21-particles were prepared from K562.mb21 cells and characterized by Nanosight and Western blot analysis. CD3-depleted PBMCs were cultured with EX21 for 14 days, NK cell amounts were monitored and media changed every 2-3 days. In vitro cytotoxicity against K562 cells were comparatively assessed for EX21-NK cells and PM21-NK cells. In vivo anti-tumor efficacy of EX21- and PM21-NK cells was assessed in NSG mice implanted ip with SKOV3_luc ovarian tumor cells (1 x 106 cells seeded for 4 days). SKOV3-bearing mice were treated with vehicle, or two doses of EX21-NK cells or PM21-NK cells (1 x 107, in 5 day intervals), and with or without in vivo administration of EX21 (10 µg, 3x/week) or PM21-particles (600 µg, 3x/week). All groups were injected ip with IL-2 (10 KU, 3x/week). Survival analysis was performed with a Log-rank (Mantel-Cox) test. Results: NK cells cultured with EX21 expanded 530 fold (344-710) over 14 days compared to 735 fold (667-802) in presence of PM21-particles. Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with either EX21 or with PM21, allowed significant ( < 0.0001) increase in survival compared to untreated animals (41-44 vs 29 days post treatment). Ip delivery of EX21 to SKOV3 bearing mice had no effect on survival in either untreated control or EX21-NK cell treated groups. Conclusions: EX21 efficiently expands NK cells and EX21-NK cells have equal anti-tumor effect as PM21-NK cells, both in vitro and in vivo.