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

Abstract

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 IL2Rgamma^null (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.

Figure 1

PD-L1 t-haNK cells are haNK cells engineered to target PD-L1-expressing tumor cells. (A) Immune-related transcriptomes of irradiated PD-L1 t-haNK and haNK cells were analyzed using the nCounter PanCancer immune profiling panel. Heatmap showing select NK-related genes with data presented as fold change values on a scale of −3 (blue) to 3 (red). (B) Representative FACS plots showing the frequencies of anti-PD-L1 CAR⁺, CD16⁺, and perforin⁺granzyme B⁺ irradiated PD-L1 t-haNK and haNK cells. Results are from two independent experiments and were gated on live cells. Blue histograms represent controls, while red histograms represent samples. (C) Immunofluorescence microscopy data showing perforin (green) and NKG2D (orange) expressions on irradiated PD-L1 t-haNK cells. (D) MDA-MB-231 tumor cell lysis by PD-L1 t-haNK and haNK cells with or without anti-PD-L1 antibody (1 µg/mL) was evaluated via ¹¹¹In-release assay at 25:1 E:T ratio. Results shown are the means with SEM of triplicate measurement and representative of three independent experiments. (E) Cytolytic capacity of PD-L1 t-haNK cells on CMA and anti-CD16 treatment with MDA-MB-231 as target at 25:1 E:T ratio as evaluated in ¹¹¹In-release assay. Results shown are the means with SEM of triplicate measurement. (F) PD-L1 t-haNK cell-mediated killing of MDA-MB-231 tracked in real time using live-cell imaging. CellTracker violet BMQC was used to differentiate the PD-L1 t-haNK cells (blue) from the MDA-MB-231 tumor cells (bright field), and CellEvent-caspase 3/7 was used to identify cells undergoing apoptosis. Scale bars on microscopy images (C, F) represent 10 µM. One-way analysis of variance with Tukey’s multiple comparison test was used for statistical analyses. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001. CAR, chimeric antigen receptor; CMA, concanamycin A; E:T, effector to target; FACS, fluorescence-activated cell sorting; haNK, high-affinity NK; NK, natural killer; PD-L1, programmed death-ligand 1; t-haNK, targeting high-affinity natural killer.

Figure 2

PD-L1 t-haNK cells induced the lysis of a range of human cancer cell lines in vitro. (A) Cell lysis of MDA-MB-231, BT549, T47D, SUM149, MCF7, H460, H441, HCC4006, SW480, SW620, DU145, HTB1, CaSKi, CH22, and IOMM mediated by PD-L1 t-haNK cells and haNK cells±anti-PD-L1 AB (1 µg) at different E:T ratios evaluated in ¹¹¹In-release assay. Results shown are the means with SEM of triplicate measurement and representative of at least two independent experiments for each cell line. (B) Correlation between PD-L1 score and % tumor cell lysis (p=0.0133, r=0.6222). The PD-L1 score of each cell line was calculated by scoring the % PD-L1⁺ cells and the MFI on a quartile scale of 1–4 and then adding the two values. The best-fit lines were determined using linear regression. E:T, effector to target; MFI, mean fluorescence intensity; PD-L1, programmed death-ligand 1; t-haNK, targeting high-affinity natural killer.

Figure 3

IFN-γ pretreatment improved PD-L1 t-haNK cell targeting of human breast cancer cell lines in vitro. MDA-MB-231, BT549, T47D, and MCF7 were pretreated overnight with IFN-γ prior to being incubated with PD-L1 t-haNK cells. Cell lysis was evaluated in ¹¹¹In-release assay at 25:1 E:T ratio. Values represent the %PD-L1 (MFI) for each cell treatment. Results shown are the means with SEM of triplicate measurement and representative of two independent experiments. One-way analysis of variance with Tukey’s multiple comparisons test was used for statistical analyses. *P<0.05, **P<0.01. E:T, effector to target; IFN, interferon; MFI, mean fluorescence intensity; PD-L1, programmed death-ligand 1; t-haNK, targeting high-affinity natural killer.

Figure 4

PD-L1 t-haNK cells preferentially targeted PD-L1^high versus PD-L1^low human breast cancer cell line in vitro. Carboxyfluorescein succinimidyl ester (CFSE)-labeled PD-L1^high MDA-MB-231 was cocultured with CellTrace Violet-labeled PD-L1^low breast cancer cell lines BT549 (A, B), MCF7 (C, D), and T47D (E, F). CellTrace Violet-labeled IFN-γ-treated MDA-MB-231 was cocultured with CFSE-labeled untreated cells (G, H). Flow-sorted PD-L1^high and PD-L1^low MDA-MB-231 cell populations were labeled with CFSE and CellTrace Violet, respectively (I, J). Irradiated PD-L1 t-haNK cells were incubated with the cocultures at different E:T(PD-L1^high):T(PD-L1^low) overnight prior to flow cytometric analysis to determine cell lysis. The flow cytometric plots shown have been stratified to live cells and have been downsampled, such that all the plots for each coculture have the same cell count in every E:T:T ratio. The numbers indicate the cell count for each population in the downsampled plots. Data are representative of two independent experiments for each cell line for panels A–F. E:T, effector to target; IFN, interferon; PD-L1, programmed death-ligand 1; t-haNK, targeting high-affinity natural killer.

Figure 5

PD-L1 silencing on MDA-MB-231 diminished the ability of PD-L1 t-haNK cells to target the tumor cells in vitro. PD-L1 null MDA-MB-231 was generated through the CRISPR/Cas9 system. PD-L1 knockout was confirmed via (A) flow cytometry, (B) western blot, and (C) immunofluorescence microscopy. Scale bar=75 µm (D). WT MDA-MB-231 and PD-L1 null MDA-MB-231 tumor cell lysis mediated by PD-L1 t-haNK cells was evaluated via ¹¹¹In-release assay at different E:T ratios. (E) WT MDA-MB-231 and PD-L1 null MDA-MB-231 tumor cell lysis mediated by PD-L1 t-haNK and haNK cells were evaluated via ¹¹¹In-release assay at 50:1 E:T ratio. Results for via ¹¹¹In-release assay shown are the means with SEM of triplicate measurement and representative of two independent experiments. (F) CFSE-labeled WT MDA-MB-231 and CellTrace Violet-labeled PD-L1 null MDA-MB-231 were cocultured together and added with irradiated PD-L1 t-haNK cells at different E:T:T ratios overnight before flow cytometric analysis of cell lysis. The FACS plots shown have been stratified to live cells and downsampled such that each plot has the same cell count in each E:T:T ratio. The numbers indicate the cell count for each population in the downsampled plots. Data are representative of two independent experiments for each cell line. Two-way analysis of variance with Sidak’s multiple comparison test was used for statistical analyses. **P<0.01, ***P=0.09, ****P<0.0001. E:T, effector to target; PD-L1, programmed death-ligand 1; CFSE, carboxyfluoresceinsuccinimidyl ester; DAPI,4′,6-diamidino-2-phenylindole; ns, not significant; t-haNK, targeting high-affinity natural killer; WT, wild type.

Figure 6

PD-L1 t-haNK cells inhibited primary and metastatic tumor growth in vivo. (A, B) Female NSG mice (10–16 weeks old) were inoculated with WT (A) or PD-L1 null MDA-MB-231 (B) tumors. The tumor-bearing mice (n=10/group) were treated with irradiated PD-L1 t-haNK cells intraperitoneally two times per week for 4 weeks and tumor growth was monitored. Data shown are representative of two independent experiments. (C, D) Macrometastases were counted from liver (C) and lung (D) tissues collected from the WT MDA-MB-231-bearing mice cohorts. (E) PD-L1 t-haNK cell and CMA-treated irradiated PD-L1 t-haNK cells were injected into MDA-MB-231 tumor-bearing female NSG mice (n=10/group) once a week for 4 weeks and tumor growth was monitored. (F, G) HTB1-bearing (F, n=7–8 mice/group) and H460-bearing (G, n=10/group) female NSG mice were treated with PD-L1 t-haNK cells once a week and tumor growth was monitored. Arrows in the growth curves indicate PD-L1 t-haNK cell treatment. Two-way analysis of variance with Tukey’s (E) or Sidak’s (A, B, F, G) multiple comparisons test statistical analyses of the tumor growth curves. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001. Student’s t-test was used for the statistical analyses of the metastatic lesions. *P<0.05. CMA, concanamycin A; NSG, NOD-scid IL2Rgamma^null; PD-L1, programmed death-ligand 1; t-haNK, targeting high-affinity natural killer; WT, wild type.

Figure 7

PD-L1 t-haNK cells, α-PD-1, and N-803 combination therapy resulted in superior tumor growth control. (A, B) PD-L1 t-haNK (A) and NK cells isolated from healthy human donor PBMCs (B) were cocultured with MDA-MB-231 cells in the presence of N803 (50 ng/mL), and target cell killing was assessed via ¹¹¹In-release assay. (C) Splenocytes from C57BL/6 mice were incubated with varying amounts of N-803 for 24 hours. Afterwards, IFN-γ levels in the culture supernatant were quantified via ELISA. (D) MOC1 cells were incubated for 24 hours in supernatants harvested from untreated or N-803-treated (1 μg) splenocyte cultures as described in panel C. As positive control, MOC1 cells were incubated with IFN-γ (20 ng/mL). The MOC1 cells were then analyzed for PD-L1 expression and used as target cells for PD-L1 t-haNK cell-mediated killing via ¹¹¹In-release assay. (E) Model of indirect enhancement in killing of tumors by N-803 mediated increase of tumor PD-L1. (F) C67BL/6 mice were transplanted with MOC1 cells. The tumor-bearing mice (n=8 mice/group) were treated starting at day 10 (tumor volume ~80 to 100 mm³) with once weekly PD-1, N-803 or PD-L1 t-haNK cells alone or in concurrent combination for a total of three treatments. The arrows below the x-axis of growth plots indicate individual or concurrent treatments. Inset under the legend for each plot is the number of established tumors that rejected with treatment. Two-way analysis of variance with Tukey’s multiple comparison test was used for statistical analyses. ***P=0.09, ****P<0.0001. IFN, interferon; MOC1, mouse oral cancer 1; NK, natural killer; PBMC, peripheral blood mononuclear cell; PD-L1, programmed death-ligand 1; t-haNK, targeting high-affinity natural killer.

Figure 8

PD-L1 t-haNK cells decreased human PBMC-derived MDSC population in vitro. (A) PBMCs from healthy HDs were cocultured with PD-L1 t-haNK cells at different E:T ratios overnight and then analyzed for different immune subset populations (n=11). (B) Healthy HD PBMCs and PD-L1 t-haNK cells (0.5:1.0 E:T ratio) were separated by a transwell insert and incubated overnight prior to flow cytometric analysis of MDSC populations. MDSCs were identified as CD11b⁺ CD33⁺ HLA-DR^low/− and were either CD14⁺ or CD15⁺. (C, D) PBMCs from patients with prostate cancer (C, n=3) and patients with HNSCC (D, n=3) were cocultured with PD-L1 t-haNK cells at different E:T ratios and were analyzed for mMDSC (CD11b⁺ CD33⁺ HLA-DR^low/− CD14⁺ CD15⁻) and gMDSC (CD11b⁺ CD33⁺ HLA-DR^low/− CD14⁻ CD15⁺) populations after an overnight coincubation. (E) MDSCs were isolated from healthy donor PBMCs using a CD33 isolation kit and were used as PD-L1 t-haNK cell targets. Cell lysis was evaluated through ¹¹¹In-release assay at 20:1 E:T ratio. **P<0.01. cDC, conventional dendritic cells; E:T, effector to target; gMDSC, granulocytic myeloid-derived suppressor cell; HD, human donor; HNSCC, head and neck squamous cell carcinoma; MDSC, myeloid-derived suppressor cell; mMDSC, monocytic myeloid-derived suppressor cell; NK, natural killer; PBMC, peripheral blood mononuclear cell; PD-L1, programmed death-ligand 1; pDC, plasmacytoid dendritic cells; t-haNK, targeting high-affinity natural killer.