Combined IL-15 and IL-12 drives the generation of CD34⁺-derived natural killer cells with superior maturation and alloreactivity potential following adoptive transfer

Abstract

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against cancer, including acute myeloid leukemia (AML). Previously, we reported a cytokine-based culture method for the generation of NK cell products with high cell number and purity. In this system, CD34⁺ hematopoietic progenitor cells (HPC) were expanded and differentiated into NK cells under stroma-free conditions in the presence of IL-15 and IL-2. We show that combining IL-15 with IL-12 drives the generation of more mature and highly functional NK cells. In particular, replacement of IL-2 by IL-12 enhanced the cytolytic activity and IFNγ production of HPC-NK cells toward cultured and primary AML cells in vitro, and improved antileukemic responses in NOD/SCID-IL2Rγnull (NSG) mice bearing human AML cells. Phenotypically, IL-12 increased the frequency of HPC-NK cells expressing NKG2A and killer immunoglobulin-like receptor (KIR), which were more responsive to target cell stimulation. In addition, NK15/12 cell products demonstrated superior maturation potential, resulting in >70% positivity for CD16 and/or KIR within 2 weeks after infusion into NSG mice. We predict that higher functionality and faster in vivo maturation will favor HPC-NK cell alloreactivity toward malignant cells in patients, making this cytokine combination an attractive strategy to generate clinical HPC-NK cell products for cancer adoptive immunotherapy.

Keywords:

• adoptive immunotherapy
• acute myeloid leukemia
• interleukin-12
• interferon-gamma
• killer immunoglobulin-like receptor
• natural killer cell

Abbreviations:

• AML, acute myeloid leukemia
• BM, bone marrow
• HPC, hematopoietic progenitor cell
• HSCT, hematopoietic stem cell transplantation
• NK, natural killer
• NSG, NOD/SCID-IL2Rγnull
• KIR, killer immunoglobulin-like receptor
• UCB, umbilical cord blood

Disclosure of Potential Conflicts of Interest

The work has been performed in collaboration with Glycostem Therapeutics, a life-science company in the Netherlands. The authors Jan Spanholtz and Marleen Tordoir are employees of Glycostem Therapeutics that worked in the laboratory of Harry Dolstra, who acted as scientific consultant. Other authors have no conflict of interest to declare.

Acknowledgments

We thank Rob Woestenenk and colleagues from the flow cytometry facility (Radboudumc) for technical support and cell sorting. We also thank Jos Paardekooper and other analysts from the laboratory of Hematology (Radboudumc), as well as the team of the animal facility.

Author Contributions

JC designed and performed experiments, analyzed data and wrote the manuscript; AQ performed experiments and analyzed data; JS and MT provided instructions for applying GBGM® expansion / differentiation technology and assisted in UCB-NK cell culture; JJ, RV, and NS provided advice; HD supervised research. All authors revised and approved the manuscript.

Funding

This work was supported by ZonMW 11600101, Vanderes Foundation 2009–2016, RUMC-RIO 2011, KWF KUN2014-6701, and the Radboudumc.