Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

Abstract

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.

NK cell-based immunotherapy for malignant diseases

“…167 Compared with mbIL15, K562-based genetically engineered artificial antigen-presenting cells with membrane-bound IL-21 supported human NK cell proliferation with longer telomeres and less senescence, resulting in enhanced expansion and tumor killing. 168 Large-scale in vitro-expanded NK cells using irradiated Epstein-Barr virustransformed lymphoblastoid cell lines feeder cells were also found to be more cytotoxic to tumor cells, with upregulated activating receptors and death receptor ligands as well as altered cytokine secretion profiles. The safety and antitumor effects of autologous NK cells expanded from PBMCs were investigated in a phase I trial in patients with advanced metastatic tumors and hematological malignancies.…”

PD-L1 blockade enhances anti-tumor efficacy of NK cells

“…Lee and co-workers have developed a method for large-scale production of highly potent NK cells using K562 cells engineered to express 4-1BB ligand and membrane bound IL21 (mb21). 22 NK cells expanded by the K562-mb21 feeder cell method are currently used in multiple clinical trials and preliminary results are showing highly encouraging clinical efficacy for leukemia relapse prevention after stem cell transplant. 15 Recently, the K562-mb21 cell based method was modified to a cell-free, PM21-particle based method for both ex vivo and in vivo specific expansion of NK cells which can eliminate some logistical and safety concerns while also retaining the benefits of the feeder-cell based expansion.…”

Extending the lifespan and efficacies of immune cells used in adoptive transfer for cancer immunotherapies–A review

“…Induces Apoptosis 107,108 Telomerase activation 122 Unconfirmed Unconfirmed IL- 15 Decrease apoptosis 136 Decrease apoptosis 130,131 Unconfirmed Telomerase activation [123][124][125] Decrease apoptosis 132 engineering of cells described here, that can be regulated by transfection with suicide genes such as those for herpes simplex virus thymidine kinase (TK) 140 or CD20 141 or the use of transfected extracellular FK506 binding domains with Caspase-9 signaling motifs to cause cell death upon treatment with FK506. 142 …”

“…121 IL-21 has also been demonstrated to increase NK cell longevity by maintaining telomere length, suggesting telomerase activity is taking place. 122 Considering CD8 C T cells, IL-15 has been shown to increase CD8 C memory T cell longevity by inducing telomerase activity through JAK3 and PI3K signaling pathways. 123,124 A further study, looking at both IL-7 and IL-15 showed an upregulation of na€ ıve CD8 C T cells when incubated with these cytokines, with a higher response seen with IL-15.…”