ADCC employing an NK cell line (haNK) expressing the high affinity CD16 allele with avelumab, an anti-PD-L1 antibody

Abstract

NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In the studies reported here, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Studies also show that while NK cells have the ability to lyse aNK or haNK cells, the addition of NK cells to irradiated haNK cells does not inhibit haNK-mediated lysis of human tumor cells, with or without the addition of avelumab. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells.

Keywords: ADCC; NK-92 cells; anti-PD-L1; avelumab; haNK cells.

Figure 1

Gene expression of haNK cells ± 10 Gy irradiation. haNK cells were intact or irradiated 24 hr prior to RNA extraction and analysis by nCounter PanCancer Immune Profiling Panel. Heat Map Euclidean Distance Analysis depicts the top differentially expressed genes by irradiation in comparison to non-irradiated haNK cells (a), nSolver analysis software version 3.0.22, (P < 0.05). Upregulated (top panel) and downregulated (bottom panel) genes are shown for two independent experiments (left and right panels). Top Upstream Regulators and Diseases and Biological Functions predicted to be associated with the corresponding upregulated (b) and downregulated (c) genes by Ingenuity Pathway Analysis are shown. Several genes were upregulated by irradiation (CD19, CD37, CTSG, CXCL8, FOS, IKBKG, IL12RB1, IL2RA, IRAK1, LAG3), while others were downregulated (ADA, C3AR1, CASP8, CTLA4, FYN, HLA-DMB, IL15, IL18R1, INPP5D, ITGAM, ITGB1, KIT, KLRB1, LILRB2, LYN, NCR1, PSEN2, PTPRC, SPP1, STAT4, TIGIT, TLR6). The NF-κB complex was predicted to be affected by irradiation as IRAK1 and IKBKG were upregulated and IL18R1 and INPP5D were downregulated. Additionally, FOS, a nuclear phosphoprotein important for the AP-1 transcription factor complex, was upregulated by irradiation. Downregulated genes include those associated with immune cell activation by the Src tyrosine kinases: FYN, LYN, and PTPRC. Downregulated genes involved in immune-regulation include CTLA4, HLA-DMB, and TIGIT, whereas LAG3 was found to be upregulated. NK cell–related genes that were downregulated include KLRB1, TIGIT and NCR1, whereas IL12RB1 was upregulated by irradiation.

Figure 2

haNK ADCC mediated by avelumab. Tumor cell lysis mediated by irradiated haNK cells and avelumab (ADCC) was evaluated in ¹¹¹In-release assays at different E:T ratios as indicated. 0 indicates target cell lysis in the absence of effector cells. Both 4-hr and 18-hr assays were performed for (a) H460 lung carcinoma, and (b) CaSki cervical carcinoma, employing avelumab (blue circles) or IgG (black squares) at 1 ng/ml. All other assays were 18 hr, and c-f used avelumab or IgG at 2 ng/ml; HCC4006: lung carcinoma; H441: lung carcinoma; SKOV3: ovarian carcinoma; MDA-MB-231: breast carcinoma. g, HTB-4: bladder carcinoma employed avelumab or IgG at 2 μg/ml. Results shown are the averages (SD) of triplicate measurements from one of at least three comparable repeat experiments. Multiple t-tests were used to compare each avelumab dose with IgG control at all E:T ratios. *** p < 0.001, **p < 0.01, * p < 0.05.

Figure 3

Characterization of haNK lysis and ADCC induced by avelumab. a: The H460 human lung carcinoma cell line was used as a target in an 18-hr assay to evaluate if haNK ADCC mediated by avelumab (1 ng/ml) could be blocked using anti-CD16 antibody (50 μg/ml). (b) The H460 cell line was used as a target in an 18-hr assay to evaluate if haNK ADCC mediated by avelumab (1 ng/ml) could be blocked using CMA. Irradiated haNK cells were used at a 25:1 E:T ratio. Results are the mean (SEM) lysis of triplicate measurements in one of four repeat experiments. Dotted lines show the percent endogenous haNK lysis. T-tests were employed to compare the treatments. (c) Irradiated haNK cells and non-irradiated NK cells from a healthy donor (HD) were evaluated for killing frequency. NK cells were co-incubated with ¹¹¹In-labeled H460 target cells at low E:T ratios for 18 h. % specific lysis was used to calculate killing frequency by dividing the number of target cells killed by the number of effector cells used for the 0.625:1 ratio. *** P< 0.001, ** P< 0.01, * P< 0.05.

Figure 4

MDA-MB-231 breast carcinoma cells (a-f), and CaSki cervical carcinoma cells (g-h) were ¹¹¹In-labeled as targets and incubated with haNK cells alone, NK cells alone, or haNK and NK cells together at ratios mimicking those seen in the blood after infusion of 10⁸ haNK cells (a-b) or 10⁹ haNK cells (c-h). haNK lysis (with IgG1 control antibody) and ADCC induced by avelumab (2 ng/ml) or cetuximab (1 μg/ml) were evaluated. Bars show the mean (SD) lysis of triplicate wells. NK cells from four different healthy donors and three cancer patients were tested with similar results, and the results from one healthy donor and one cancer patient are shown. Additional results are shown in Supplemental Figure 2. (a-b) Using MDA-MB-231 cells as targets, haNK cells were used at a 5:1 E:T ratio (blue), NK cells were used at a 50:1 E:T ratio (white), and NK cells and haNK cells were combined at a 10:1 ratio, resulting in an overall E:T ratio of 55:1 (grey) to mimic NK:haNK ratios in the blood after infusion of 10⁸ haNK cells to a 70 kg patient. (c-f) Using MDA-MB-231 cells as targets, haNK cells were used at a 5:1 E:T ratio (blue), NK cells were used at a5:1 E:T ratio (white), and NK cells and haNK cells were combined at a 1:1 ratio, resulting in an overall E:T ratio of 10:1 (grey) to mimic NK:haNK ratios in the blood after infusion of 10⁹ haNK cells to a 70 kg patient, showing the results for the healthy donor in c and d, and the results for the cancer patient in e and f. (g-h) An additional tumor cell line, CaSki, was used as a target with NK cells from the cancer patient as effectors. haNK cells were used at a 5:1 E:T ratio (blue), NK cells were used at a 5:1 E:T ratio (white), and NK cells and haNK cells were combined at a 1:1 ratio, resulting in an overall E:T ratio of 10:1 (grey) to mimic NK:haNK ratios in the blood after infusion of 10⁹ haNK cells to a 70 kg patient.

Figure 5

Avelumab alone does not lyse tumor cells, and haNK cells have no cytotoxic activity against other haNK cells. (a) Irradiated haNK cells were used in an 18-hr assay, using MDA-MB-231 (human breast carcinoma) as a target at an E:T ratio of 7.5:1. Target cells were also incubated with avelumab (2 ng/ml) or isotype control IgG1 Ab (2 ng/ml) alone, without effector cells. haNK killing (black bars) and ADCC mediated by avelumab (grey bars) are shown. (b) Irradiated haNK cells were used in a 4-hr assay, using irradiated haNK cells or MDA-MB-231 cells as targets at an E:T ratio of 20:1. Endogenous haNK lysis (black bars) and ADCC mediated by avelumab (2 μg/ml) (grey bars) are shown.