There is a great interest in developing natural killer (NK) cells as adoptive cancer immunotherapy. For off-the-shelf approaches and to conduct multicenter clinical trials, cryopreserved NK cells are the preferred product. However, recent studies reported that cryopreservation of NK cells results in loss of cell motility and, as a consequence, cytotoxicity which limits the clinical utility of such products. This study assessed the impact of cryopreservation on the recovery and function of PM21-particle expanded NK cells (PM21-NK cells) as well as their antitumor activity in vitro using 2D and 3D cancer models and in vivo in ovarian cancer models, including patient-derived xenografts (PDX). Viable PM21-NK cells were consistently recovered from cryopreservation and overnight rest with a mean recovery of 73 ± 22% (N = 19). Thawed and rested NK cells maintained the expression of activating receptors when compared to expansion-matched fresh NK cells. Cryopreserved NK cells that were thawed and rested showed no decrease in cytotoxicity when co-incubated with tumor cells at varying effector-to-target (NK:T) ratios compared to expansion-matched fresh NK cells. Moreover, no differences in cytotoxicity were observed between expansion-matched cryopreserved and fresh NK cells in 3D models of tumor killing. These were analyzed by kinetic, live-cell imaging assays co-incubating NK cells with tumor spheroids. When exposed to tumor cells, or upon cytokine stimulation, cryopreserved NK cells that were thawed and rested showed no significant differences in surface expression of degranulation marker CD107a or intracellular expression of TNFα and IFNγ. In vivo antitumor activity was also assessed by measuring the extension of survival of SKOV-3-bearing NSG mice treated with fresh vs. cryopreserved NK cells. Cryopreserved NK cells caused a statistically significant survival extension of SKOV-3-bearing NSG mice that was comparable to that observed with fresh NK cells. Additionally, treatment of NSG mice bearing PDX tumor with cryopreserved PM21-NK cells resulted in nearly doubling of survival compared to untreated mice. These data suggest that PM21-NK cells can be cryopreserved and recovered efficiently without appreciable loss of viability or activity while retaining effector function both in vitro and in vivo. These findings support the use of cryopreserved PM21-NK cells as a cancer immunotherapy treatment.

Treatments targeting TIGIT have gained a lot of attention due to strong preclinical and early clinical results, particularly with anti-PD-(L)1 therapeutics. However, this combination has failed to meet progression-free survival endpoints in phase III trials. Most of our understanding of TIGIT comes from studies of T cell function. Yet, this inhibitory receptor is often upregulated to the same, or higher, extent on NK cells in cancers. Studies in murine models have demonstrated that TIGIT inhibits NK cells and promotes exhaustion, with its effects on tumor control also being dependent on NK cells. However, there are limited studies assessing the role of TIGIT on the function of human NK cells (hNK), particularly in lung cancer. Most studies used NK cell lines or tested TIGIT blockade to reactivate exhausted cells obtained from cancer patients. For therapeutic advancement, a better understanding of TIGIT in the context of activated hNK cells is crucial, which is different than exhausted NK cells, and critical in the context of adoptive NK cell therapeutics that may be combined with TIGIT blockade. In this study, the effect of TIGIT blockade on the anti-tumor activities of human ex vivo-expanded NK cells was evaluated in vitro in the context of lung cancer. TIGIT expression was higher on activated and/or expanded NK cells compared to resting NK cells. More TIGIT+ NK cells expressed major activating receptors and exerted anti-tumor response as compared to TIGIT− cells, indicating that NK cells with greater anti-tumor function express more TIGIT. However, long-term TIGIT engagement upon exposure to PVR+ tumors downregulated the cytotoxic function of expanded NK cells while the inclusion of TIGIT blockade increased cytotoxicity, restored the effector functions against PVR-positive targets, and upregulated immune inflammation-related gene sets. These combined results indicate that TIGIT blockade can preserve the activation state of NK cells during exposure to PVR+ tumors. These results support the notion that a functional NK cell compartment is critical for anti-tumor response and anti-TIGIT/adoptive NK cell combinations have the potential to improve outcomes.

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In this study we propose to circumvent HLA-E/NKG2A mediated Natural Killer (NK) cell inhibition by suppression of NKG2A, to enhance cytotoxicity of ex vivo expanded NK cells. One mechanism employed by tumor cells to evade immunosurveillance is through induction of the surface expression of unconventional HLA ligands that agonize inhibitory receptors on immune cells. Specifically on some tumors, HLA-E is either expressed or its expression is known to be induced by IFNγ and is indicative of resistance to immunotherapy. HLA-E agonizes the CD94/NKG2A inhibitory complex on NK cells and some T cells to lessen their cytotoxicity, potentially decreasing the efficacy of cellular therapy with these immune cells. The CD94/NKG2A inhibitory complex is in balance with CD94/NKG2C stimulatory complex. HLA-E binds both of these complexes and the cytolytic activity of NK cells is influenced by the relative ratios of CD94 complexed to NKG2A or NKG2C. Thus, altering the ratio to increase NKG2C will increase NK cell cytotoxicity. In this study we show that NK cells ex vivo expanded with PM21-particle technology are highly cytotoxic and have elevated expression of NKG2A. These expanded NK cells also secrete high levels of IFNγ, inducing expression of HLA-E in tumor cells. To increase the cytotoxic potential of these ex vivo expanded NK cells, the potential to form CD94/NKG2A inhibitory complex was suppressed by either antibody blockade of NKG2A or deletion of the NKG2A gene by CRISPR/Cas9 editing. Cytotoxicity of NK cells against a lung cancer cell line stably expressing HLA-E were determined by kinetic live-cell imaging. Multiple NK cell:target (NK:T) ratios were used to show an increase in cytotoxicity over control NK cell conditions (alone or with isotype control) in the presence of NKG2A blocking antibodies with 39% increase observed at 1:10 NK:T ratio at 96h with anti-NKG2A and 56% increase with NKG2A knockout NK cells. These data suggest that blockade of CD94/NKG2A complex can improve cytotoxic activity of ex vivo expanded NK cells and could provide a promising effector population with the potential for enhanced therapeutic efficacy. Citation Format: Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Thomas A. Dieffenthaller, Alicja J. Copik. Suppression of NKG2A mediated inhibition in ex vivo expanded natural killer cells increases their cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2819.