A Fusion Protein Complex that Combines IL-12, IL-15, and IL-18 Signaling to Induce Memory-Like NK Cells for Cancer Immunotherapy

Cancer Immunol Res. 2021 Sep;9(9):1071-1087. doi: 10.1158/2326-6066.CIR-20-1002. Epub 2021 Jul 9.


Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Humans
  • Immunologic Memory / drug effects
  • Interleukin-12 / pharmacology*
  • Interleukin-15 / pharmacology*
  • Interleukin-18 / pharmacology*
  • Killer Cells, Natural / immunology*
  • Leukemia / immunology
  • Leukemia / therapy*
  • Mice
  • Receptors, Natural Killer Cell / metabolism
  • Recombinant Fusion Proteins / pharmacology
  • Remission Induction
  • Xenograft Model Antitumor Assays


  • Interleukin-15
  • Interleukin-18
  • Receptors, Natural Killer Cell
  • Recombinant Fusion Proteins
  • Interleukin-12