IL-21 Enhances Natural Killer Cell Response to Cetuximab-Coated Pancreatic Tumor Cells

Abstract

Purpose: Alternative strategies to EGFR blockage by mAbs is necessary to improve the efficacy of therapy in patients with locally advanced or metastatic pancreatic cancer. One such strategy includes the use of NK cells to clear cetuximab-coated tumor cells, as need for novel therapeutic approaches to enhance the efficacy of cetuximab is evident. We show that IL-21 enhances NK cell-mediated effector functions against cetuximab-coated pancreatic tumor cells irrespective of KRAS mutation status.

Experimental design: NK cells from normal donors or donors with pancreatic cancer were used to assess ADCC, IFN-γ release, and T-cell chemotaxis toward human pancreatic cancer cell lines. The in vivo efficacy of IL-21 in combination with cetuximab was evaluated in a subcutaneous and intraperitoneal model of pancreatic cancer.

Results: NK cell lysis of cetuximab-coated wild-type and mutant kras pancreatic cancer cell lines were significantly higher following NK cell IL-21 treatment. In response to cetuximab-coated pancreatic tumor cells, IL-21-treated NK cells secreted significantly higher levels of IFN-γ and chemokines, increased chemotaxis of T cells, and enhanced NK cell signal transduction via activation of ERK and STAT1. Treatment of mice bearing subcutaneous or intraperitoneal EGFR-positive pancreatic tumor xenografts with mIL-21 and cetuximab led to significant inhibition of tumor growth, a result further enhanced by the addition of gemcitabine.

Conclusions: These results suggest that cetuximab treatment in combination with IL-21 adjuvant therapy in patients with EGFR-positive pancreatic cancer results in significant NK cell activation, irrespective of KRAS mutation status, and may be a potential therapeutic strategy. Clin Cancer Res; 23(2); 489-502. ©2016 AACR.

Figure 1. Expression of EGFR in human pancreatic cancer cell lines

(A) Lysates were prepared from human pancreatic cancer cell lines and subjected to immunoblot analysis with antibodies directed against EGFR. Nitrocellulose membranes were probed for β-actin to control for loading. HeLa and MDA-MB-453 served as positive and negative controls, respectively. (B) The percentage of cells expressing EGFR was assessed by flow cytometry using an anti-EGFR-PE Ab. Experiments are representative of two separate determinations.

Figure 2. IL-21 enhances antibody-dependent cellular cytotoxicity of NK cells against pancreatic cancer cells

Purified human NK cells were incubated overnight in medium alone or in medium supplemented with 10 ng/ml IL-21. The lytic activity of IL-21-activated NK cells was then assessed in a standard 4 hr chromium release assay using cetuximab-coated KRAS wild-type (A) or KRAS mutant (B–D) pancreatic cancer cells as targets. (E) Graphical summary of cytotoxicity data of four pancreatic cancer cell lines at the 50:1 E:T ratio. ^‡Denotes presence of a KRAS mutation. The percentage of lysis was calculated as previously described. Each graph depicts the results from one representative donor ± SD. Three normal donors were tested per cell line. The asterisk (*) denotes p<0.001 versus all conditions shown.

Figure 3. IL-21 enhances antibody-dependent cellular cytotoxicity of pancreatic patient NK cells against KRAS mutant pancreatic cancer cells

Purified NK cells from patients with pancreatic adenocarcinoma were incubated overnight with medium alone or in medium supplemented with 10 ng/ml IL-21. The lytic activity of IL-21-activated NK cells was then assessed in a standard 4 hr chromium release assay using cetuximab-coated KRAS mutant AsPc1 tumor cells. The mean percent lysis at 25:1 E:T ratio for 9 patients who received neoadjuvant chemotherapy (A) or the 8 patients who had not received chemotherapy prior to surgical resection (B) is shown in box plots. The box plots represent the median and interquartile range, with I bars showing the range for each group. (C) NK cell lysis of a patient that underwent neuroendocrine tumor resection ± SD. Experiments are representative of two separate determinations. The asterisk (*) denotes p<0.001 versus all conditions shown.

Figure 4. Human NK cells secrete high levels of IFN-γ and NK culture supernatants stimulate the migration of T cells

The KRAS wild-type BxPc3 (A) and KRAS mutant AsPc1 and MiaPaCa2 (B) cell lines were cultured with human NK cells in an in vitro tumor co-culture assay. Control conditions consisted of tumor cells and NK cells cultured with medium alone, IgG alone, cetuximab alone, IL-21 alone or the combination of IgG and IL-21. Culture supernatants were harvested at 48 hrs and analyzed for IFN-γ by ELISA. (C) Chemotaxis of activated T cells in response to culture supernatants derived from NK cells costimulated with cetuximab-coated KRAS wild-type BxPc3 or KRAS mutant AsPc1 tumor cells and IL-21. Culture medium supplemented with monokine induced by gamma-interfereon (MIG) was included as a positive control. Each graph depicts the results from one representative donor ± SD. Three normal donors were tested per cell line. The asterisk (*) denotes p<0.001 versus all conditions shown.

Figure 5. MDSCs inhibit NK cell IFN-γ production and ADCC

Autologous PBMC-derived MDSCs or control PBMCs were co-cultured with healthy donor NK cells at a 0.5:1 ratio. In vitro generated MDSCs were found to be suppressive to T cell proliferation by CFSE assay (data not shown). (A) Purified IL-21-activated NK cells were co-cultured with cetuximab-coated KRAS mutant AsPc1 cancer cells alone or in the presence of autologous PBMCs or MDSCs. Control conditions consisted of medium alone, IL-21 alone, or cetuximab alone. Culture supernatants were harvested at 48 hrs and analyzed for IFN-γ by ELISA. (B) Purified human NK cells were incubated overnight in medium alone in medium containing 10 ng/ml of IL-21. The lytic activity was tested against cetuximab-coated KRAS mutant AsPc1 cancer cells in the presence of autologous PBMCs or MDSCs in a standard 4 hr chromium release assay. Each graph depicts the results from one representative donor ± SD. Three normal donors were tested per cell line. The asterisk (*) denotes p<0.001 versus all conditions shown.

Figure 6. NK cell signal transduction is enhanced by IL-21 receptor and Fc receptor engagement by cetuximab-coated mutant KRAS AsPc1 tumor cells

Purified human NK cells were stimulated with 10 ng/ml of IL-21 and co-cultured with cetuximab-coated KRAS mutant AsPc1 pancreatic cancer cells for 30 mins. Control conditions consisted of media alone, IL-21-stimulated NK cells alone, or cetuximab-coated tumor cells alone. Following incubation, cells were collected and underwent dual flow cytometry staining using anti-CD56 and (A) anti-phospho-ERK1/2 or (B) anti-phospho-STAT1 antibodies. Percentages reported are of dual positive populations (Q2). Each plot depicts the results from one representative donor. Three normal donors were tested per cell line.

Figure 7. The combination of IL-21 and cetuximab enhances tumor regression in a mouse xenograft model

(A) Nude mice were inoculated s.c. with 1×10⁶ Panc02^EGFR tumor cells. Once tumors reached 50–100 mm³ in size, treatment began thrice weekly with i.p. injections of PBS, 5 μg IL-21, 0.5 mg/kg cetuximab, or the combination. Tumor growth was measured by calipers thrice weekly and tumor volumes were calculated as described in the Methods section. Results shown are average tumor volumes of n=7 mice per group ± SD. The asterisk (*) denotes p<0.01 versus all conditions shown. (B) Nude mice were inoculated i.p. with 5×10⁵ Panc02^EGFR/Luc+GFP tumor cells. Treatment began on day 3 and was administered as described above. Mice were anesthetized on days 3, 11, and 17 using isoflurane. Approximately 10 min post luciferin injection, mice were imaged at an exposure time of 1 sec. (C) Xenogen IVIS software was used to quantify nonsaturated bioluminescence in regions of interest. Radiance greater than 5.5×10⁶ p/s/cm²/sr was assumed to be indicative of viable luciferase-labeled tumor cells while emissions below this range were considered as background. Results shown are average radiance of n=5 mice per group ± SD. The asterisk (*) denotes p<0.001 versus all conditions shown. (D) Nude mice bearing Panc02^EGFR tumors were treated i.p. with low dose gemcitabine (15 mg/kg) and cetuximab (0.5 mg/kg) with or without mIL-21 (5 μg) thrice weekly. Tumor volume was calculated as described above. Results shown are average tumor volumes of n=7 mice per group ± SD. The asterisk (*) denotes p<0.007 versus control condition shown. (E) Nude mice bearing Panc02^EGFR tumors were depleted of NK cells by an i.p. injection of anti-asialo GM1 or mock depleted by an i.p. injection of normal rabbit serum and treated with PBS or the combination of IL-21 and cetuximab. Results shown are average tumor volumes of n=5 mice per group ± SD. The asterisk (*) denotes p<0.002 versus control condition shown.