Cryopreserved PM21-Particle-Expanded Natural Killer Cells Maintain Cytotoxicity and Effector Functions In Vitro and In Vivo

Abstract

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.

Physicochemical Mechanisms of Protection Offered by Agarose Encapsulation during Cryopreservation of Mammalian Cells in the Absence of Membrane-Penetrating Cryoprotectants

“…Use of DMSO as a cryopreservation agent especially for infusible therapeutic cell preservation presents problems: Even though the immediate post-thaw viability of NK cells is generally reported to be quite high (albeit with some cell loss), a drastic decrease in cytotoxicity (down to 10–25% of the fresh cells) post-thaw is very common. − Furthermore, in vivo persistence of frozen–thawed NK cells is also reported to be orders of magnitude lower than fresh cells . The main reason for cytotoxicity loss is ascribed to the loss of motility, and significant changes in NK cell phenotype specifically decrease in the cytotoxic CD16+ populations. ,, To resolve the issues associated with cell number and cytotoxicity loss, therapeutic cells need to be expanded for 3–7 days post-thaw to increase cell numbers and activated through interleukin stimulation for a minimum of 12–16 h to recover cytotoxic capability. , Extra processing was required in the clinic, and the need for additional quality control/lot testing requirements increases costs, delays the treatment, and also increases the risk for contamination during expansion of the cells. Another problem with the use of DMSO in cryopreservation formulations is the patient morbidity risk, , requiring the infusion rates to be slowed and sometimes distributed over multiple sessions.…”

“…18,20,72 To resolve the issues associated with cell number and cytotoxicity loss, therapeutic cells need to be expanded for 3−7 days postthaw to increase cell numbers and activated through interleukin stimulation for a minimum of 12−16 h to recover cytotoxic capability. 19,21 Extra processing was required in the clinic, and the need for additional quality control/lot testing requirements increases costs, delays the treatment, and also increases the risk for contamination during expansion of the cells. Another problem with the use of DMSO in cryopreservation formulations is the patient morbidity risk, 73,74 requiring the infusion rates to be slowed and sometimes distributed over multiple sessions.…”

“…However, there is a pressing need to eliminate DMSO from cryopreservation protocols, especially when it is used for the stabilization of cellular therapy products. DMSO is known to adversely affect the post-thaw cytotoxic functions of natural killer (NK) cells and T-cells − while introducing significant workflow/use challenges in the clinic. ,, Many studies therefore focused on reducing the amount of DMSO needed in the cryoprotectant formulations ,− using additives such as albumin, ,, FCS/FBS, , and growth media . However, there is an ongoing debate on the safety and ethicality of using FCS/FBS as they are not clinical reagents that can be safely used in injectable/infusible cell therapy products.…”

DeepIFC: Virtual fluorescent labeling of blood cells in imaging flow cytometry data with deep learning

“…Interestingly, NK cells were better clustered by DeepIFC when fresh sample data was included in training. It has been found that NK cells may not preserve viability and may lose cytotoxicity after the freeze–thaw process [29]. This effect may be reflected in the loss of distinct NK cell morphology, leading to poor predictive performance in frozen samples.…”

Human Natural Killer Cells Cryopreserved without DMSO Sustain Robust Effector Responses

“…These protocols have unveiled the importance of cytokines such as IL-2 or IL-15 to the post-thaw stimulation of NK cells, 7−9 the possibility of precomplexing engager antibodies to retain NK cell postthaw activity, 10 and the potential of using IL-21-expressing vesicles to expand and freeze NK cells. 11 DMSO has been the cryoprotective agent (CPA) of choice, owing in large part to its ability to penetrate the cell membrane and reduce intracellular ice formation and osmotic stress during freezing and thawing. However, while effective and ubiquitous, DMSO has also not been free of controversy: it has been described as not inert, able to induce epigenetic changes in immune cells, 12 and its use has been associated with mild-tosevere side effects in patients receiving cell therapy infusions, 13−15 with direct effects on NK cell function having been reported due to its use.…”