CD137 (4-1BB) Engagement Fine-Tunes Synergistic IL-15– and IL-21–Driven NK Cell Proliferation

Abstract

To understand and dissect the mechanisms driving human NK cell proliferation, we exploited the methodology used in cell therapy to numerically expand NK cells in the presence of K562-derived artificial APC (aAPCs) and cytokines. For four consecutive weeks, high expression of CD137L by a K562-derived aAPC cell line could sustain NK cell expansion by 3 3 10 5 -fold, whereas low expression of CD137L by the parental K562 cell line only supported the expansion by 2 3 10 3 -fold. The level of expression of CD137L, however, did not modulate the sensitivity of K562 cells to the intrinsic cytotoxicity of NK cells. Similarly, the low NK cell proliferation in the presence of the parental K562 cell line and cytokines was increased by adding agonistic anti-CD137 Abs to levels similar to CD137L-expressing K562-derived aAPCs. Finally, synergy between IL-15 and IL-21 was observed only upon CD137 engagement and the presence of aAPCs. Therefore, we conclude that NK cell proliferation requires cell-to-cell contact, activation of the CD137 axis, and presence of IL-15 (or its membranous form) and IL-21. By analogy with the three-signal model required to activate T cells, we speculate that the cell-to-cell contact represents "signal 1," CD137 represents "signal 2," and cytokines represent "signal 3." The precise nature of signal 1 remains to be defined.

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

“…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]. Combining K562-4-1BBL with IL-15 and IL-21 support has resulted in superior expansion, reaching up to a 10 5 fold expansion in 4 weeks, which has been attributed to the IL-21-induced increase in telomere length, prolonging the proliferative capacity of NK cells [137].…”

“…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]. Combining K562-4-1BBL with IL-15 and IL-21 support has resulted in superior expansion, reaching up to a 10 5 fold expansion in 4 weeks, which has been attributed to the IL-21-induced increase in telomere length, prolonging the proliferative capacity of NK cells [137]. Furthermore, the activation of both STAT5 and STAT3 appears to be beneficial in proliferating NK cells.…”

Cellular therapy: Adoptive immunotherapy with expanded natural killer cells

“…The feeder cell method using K562 transduced with mbIL15 was patented and is now incorporated in the manufacture of cell products now in clinical trials. As mentioned above, the mbIL21‐expressing feeder cell concept is considered public domain, although the use of the original lines developed were licensed to Intrexon and similar engineered cell lines have been developed for NK cells manufactured by Fate Therapeutics, CytomX, Celularity, and Sanofi …”

“…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, [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]…”

“…As mentioned above, the mbIL21-expressing feeder cell concept is considered public domain, although the use of the original lines developed were licensed to Intrexon and similar engineered cell lines have been developed for NK cells manufactured by Fate Therapeutics, CytomX, Celularity, and Sanofi. 79 A variation in this approach has proven successful in which the feeder cell is manipulated to generate small (100-200 nm) plasma membrane fragments which contain the full complement of natural surface ligands and transgenes necessary to induce activation and proliferation. 68,148 This approach carries advantages of mass production and storage and improved safety by avoiding intact leukemic feeder cells in the culture process and DNA fragments in the formulated drug product.…”