Cryopreservation impairs cytotoxicity and migration of NK cells in 3-D tissue: Implications for cancer immunotherapy

Abstract

Running title: NK cell motility and cytotoxicity in a 3-D environmentSynopsis: Cryopreservation of natural killer (NK) cells dramatically impairs their motility and cytotoxicity in tissue. This finding may explain the persistent failure of clinical trials in which NK cell therapy is used for treating solid tumors. AbstractNatural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation for clinical use, the cells are cryopreserved to bridge the necessary evaluation time. While a degranulation assay confirms the ability of cryopreserved NK cells to kill target cells, we find a significant decrease of cytotoxicity after cryopreservation in a chromium release assay. We complement these standard assays with measurements of NK cell motility and cytotoxicity in 3-dimensional (3-D) collagen gels that serve as a substitute for connective tissue. We find a 5.6 fold decrease of cytotoxicity after cryopreservation and establish that this is mainly caused by a 6-fold decrease in the fraction of motile NK cells. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.

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.…”

“…18−21 Furthermore, in vivo persistence of frozen−thawed NK cells is also reported to be orders of magnitude lower than fresh cells. 71 The main reason for cytotoxicity loss is ascribed to the loss of motility, 18 and significant changes in NK cell phenotype specifically decrease in the cytotoxic CD16+ populations. 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.…”

“…71 The main reason for cytotoxicity loss is ascribed to the loss of motility, 18 and significant changes in NK cell phenotype specifically decrease in the cytotoxic CD16+ populations. 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.…”

“…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.…”

Genome engineering of induced pluripotent stem cells to manufacture natural killer cell therapies

“…Additionally, isolation from peripheral blood lymphocytes often results in a heterogenous population of lymphocytes with only a 10-20% yield of NK cells [55]. The use of primary NK cells is further complicated by their decrease in cytotoxicity following cryopreservation [56,57]. While the low yield and loss in cytotoxicity from cryopreservation can be partially overcome with cytokine support and feeder cell expansion, this approach makes PB-NK cells suboptimal for an "off-the-shelf" therapy [58][59][60].…”