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

Abstract

BackgroundAlthough 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.MethodsNanoString, 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 and111In-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.ResultsWe 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.ConclusionThese 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.

Natural Killer Cells in Immunotherapy: Are We Nearly There?

“…Tumor-associated neutrophils repress NK cell cytotoxicity through the PD-1/PD-L1 axis, and their depletion combined with PD-L1 blockade was significantly better in reducing tumor burden than either monotherapy [46]. Moreover, elimination of MDSCs with PD-L1-directed CAR-NK cells was a highly effective therapy in a MOC1 murine tumor model when combined with an anti-PD-1 antibody and the IL-15 super-agonist N-803 [47].…”

“…Tumor-associated neutrophils, which repress NK cell cytotoxicity through the PD-1/PD-L1 axis [46], and MDSCs are both potential target cells for checkpoint therapy. Indeed, CAR-NK cells that target PD-L1-expressing cells efficiently eliminate MDSCs in vitro [47]. Clinical trials that combine NK cells with monoclonal antibodies are summarized in Table 2.…”

“…For example, pharmacological inhibition of Vps34, a kinase involved in autophagy, causes the microenvironment of melanoma and colorectal cancer tumors to become more pro-inflammatory, leading to enhanced recruitment of NK cells and other immune cells, and ultimately more effective anti-PD-1/PD-L1 therapy [143]. An IL-15 superagonist, named N-803 (formerly ALT-803), which has seen widespread success as an immunotherapeutic option for multiple cancers [144,145], is being tested in a phase II clinical trial (NCT03853317) in combination with adoptive CAR-NK cells targeting PD-L1 after promising results in pre-clinical studies [47]. Interestingly, a novel molecule comprising N-803 and anti-PD-L1 domains has shown promise for different carcinoma models [144].…”

Natural Born Killers: NK Cells in Cancer Therapy

“…Due to the specific expression of PD-L1 on tumor and immunosuppressive cells, PD-L1 was identified as an optimal target for CAR therapies. A novel NK-92 cell line was engineered to express a CAR specific for PD-L1, ER-retained IL-2 and high affinity CD16, termed PD-L1 targeted-haNK (t-haNK) [ 88 ]. Exciting preclinical data demonstrated PD-L1 specific efficacy of these cells against 15 tumor cell lines in vitro, and strong anti-tumor efficacy in vivo of triple negative breast cancer, bladder and lung tumors [ 88 ].…”

The Importance of Cellular Immunity in the Development of Vaccines and Therapeutics for COVID-19

“…Multiple prior studies have used this assay to detect the dynamics of 123 immune cell subsets, many of which have known biologic functions. Recently, additional immune cell subsets have been identified as having biologic functions [ 3 ]. The most recent assay being used can now simultaneously analyze 138 immune cell subsets.…”