Expansion of Human NK Cells Using K562 Cells Expressing OX40 Ligand and Short Exposure to IL-21

Abstract

Background: Natural Killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. Here, we report a new effective method to expand human NK cells ex vivo using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in combination with a short exposure to soluble IL-21. In addition, we describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction. Methods: K562-OX40L cells were generated by lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Soluble IL-21 was also added in various concentrations only once at the beginning of the culture. NK cells were expanded for 4–5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, CD107a degranulation) of these expanded NK cells were compared to those generated by using K562 feeder cells. Results: The culture of NK cells with K562-OX40L cells in combination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2,000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis between the feeder cells and NK cells as well as the homotypic interaction between NK cells through the OX40-OX40L axis were both necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and CD107 degranulation. Conclusion: Our data suggest that OX40 ligand is a potent co-stimulant for the robust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.

You Have Got a Fast CAR: Chimeric Antigen Receptor NK Cells in Cancer Therapy

“…Genetically engineered feeder cells expressing specific surface ligands to aid in NK cell expansion have been developed. These include membrane-bound cytokines, such as IL-15, IL-18, and IL-21, as well as ligands to activating receptors expressed on NK cells, such as 4-1BBL, HLA-E, and OX40L [132,[134][135][136]. While HLA-E-expressing-modified 721.221 cells (721.221-AEH) have been used as a method to preferentially expand adaptive NKG2C + CD56 dim NK cells from a bulk NK cell population, 4-1BBL-expressing K562 cells have been used to generally increase NK cell expansion and increase the viability [132,136,137].…”

Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions

“…This is a time-consuming process as only 10% of PBMCs are NK cells [145]. However, recently developed methods are being used to enhance NK-cell expansion, such as through K562-feeder cell expression of OX40 ligand [146]. As mentioned above, a limitation to CAR-NK therapy is the extreme sensitivity of NK cells to cryopreservation.…”

Cellular therapy: Adoptive immunotherapy with expanded natural killer cells

“…In collaboration with CytoSen Therapeutics (recently acquired by Kiadis Pharma NV), we re‐derived and validated clone CSTX002 and its MCB, which is now being shared academically and is supporting NK cell propagation for several clinical trials. Similar versions have been developed and published by others, and feeder cells with soluble IL‐21 have also been used, but appear much less effective …”

“…Similar versions have been developed and published by others, [77][78][79] and feeder cells with soluble IL-21 have also been used, but appear much less effective. [80][81][82][83][84][85][86][87]…”