PD-L1 targeting high-affinity NK (t-haNK) cells induce direct antitumor effects and target suppressive MDSC populations

J Immunother Cancer. 2020 May;8(1):e000450. doi: 10.1136/jitc-2019-000450.


Background: Although immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor cells that express immune checkpoint molecules should be developed. For the first time, we report a novel natural killer (NK) cell line, programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK), which was derived from NK-92 and was engineered to express high-affinity CD16, endoplasmic reticulum-retained interleukin (IL)-2, and a PD-L1-specific chimeric antigen receptor (CAR). We show that PD-L1 t-haNK cells also retained the expression of native NK receptors and carried a high content of granzyme and perforin granules.

Methods: NanoString, flow cytometry, and immunofluorescence analyses were performed to characterize the phenotype of irradiated PD-L1 t-haNK cells. In vitro PD-L1 t-haNK cell activity against cancer cell lines and human peripheral blood mononuclear cells (PBMCs) was determined via flow-based and 111In-release killing assays. The antitumor effect of PD-L1 t-haNK cells in vivo was investigated using MDA-MB-231, H460, and HTB1 xenograft models in NOD-scid IL2Rgammanull (NSG) mice. Additionally, the antitumor effect of PD-L1 t-haNK cells, in combination with anti-PD-1 and N-803, an IL-15 superagonist, was evaluated using mouse oral cancer 1 syngeneic model in C57BL/6 mice.

Results: We show that PD-L1 t-haNK cells expressed PD-L1-targeting CAR and CD16, retained the expression of native NK receptors, and carried a high content of granzyme and perforin granules. In vitro, we demonstrate the ability of irradiated PD-L1 t-haNK cells to lyse 20 of the 20 human cancer cell lines tested, including triple negative breast cancer (TNBC) and lung, urogenital, and gastric cancer cells. The cytotoxicity of PD-L1 t-haNK cells was correlated to the PD-L1 expression of the tumor targets and can be improved by pretreating the targets with interferon (IFN)-γ. In vivo, irradiated PD-L1 t-haNK cells inhibited the growth of engrafted TNBC and lung and bladder tumors in NSG mice. The combination of PD-L1 t-haNK cells with N-803 and anti-PD-1 antibody resulted in superior tumor growth control of engrafted oral cavity squamous carcinoma tumors in C57BL/6 mice. In addition, when cocultured with human PBMCs, PD-L1 t-haNK cells preferentially lysed the myeloid-derived suppressor cell population but not other immune cell types.

Conclusion: These studies demonstrate the antitumor efficacy of PD-L1 t-haNK cells and provide a rationale for the potential use of these cells in clinical studies.

Keywords: immunology; oncology; tumors.

Publication types

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

MeSH terms

  • Animals
  • B7-H1 Antigen / antagonists & inhibitors*
  • B7-H1 Antigen / immunology
  • Cell Line, Tumor
  • Combined Modality Therapy / methods
  • Female
  • GPI-Linked Proteins / genetics
  • GPI-Linked Proteins / immunology
  • GPI-Linked Proteins / metabolism
  • Humans
  • Immune Checkpoint Inhibitors / pharmacology
  • Immune Checkpoint Inhibitors / therapeutic use
  • Immunotherapy, Adoptive / methods*
  • Interleukin-2 / genetics
  • Interleukin-2 / immunology
  • Interleukin-2 / metabolism
  • Killer Cells, Natural / immunology
  • Killer Cells, Natural / metabolism
  • Killer Cells, Natural / transplantation*
  • Mice
  • Myeloid-Derived Suppressor Cells / drug effects
  • Myeloid-Derived Suppressor Cells / immunology*
  • Neoplasms / immunology
  • Neoplasms / pathology
  • Neoplasms / therapy*
  • Programmed Cell Death 1 Receptor / antagonists & inhibitors
  • Programmed Cell Death 1 Receptor / immunology
  • Protein Engineering
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / immunology
  • Receptors, Chimeric Antigen / metabolism
  • Receptors, IgG / genetics
  • Receptors, IgG / immunology
  • Receptors, IgG / metabolism
  • Tumor Escape / drug effects
  • Tumor Escape / immunology
  • Xenograft Model Antitumor Assays


  • B7-H1 Antigen
  • CD274 protein, human
  • FCGR3B protein, human
  • GPI-Linked Proteins
  • IL2 protein, human
  • Immune Checkpoint Inhibitors
  • Interleukin-2
  • PDCD1 protein, human
  • Programmed Cell Death 1 Receptor
  • Receptors, Chimeric Antigen
  • Receptors, IgG