Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy

Abstract

AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC. The AXL role in oncogenesis was studied using the short hairpin RNA approach in a pancreatic carcinoma cell line. We further generated antihuman AXL mAbs and evaluated their inhibitory effects and the AXL downstream signaling pathways first in vitro, in a panel of pancreatic cancer cell lines and then in vivo, using subcutaneous or orthotopic pancreatic tumor xenografts. AXL receptor was found expressed in 76% (32/42) of PDAC and was predominantly present in invasive cells. The AXL-knockdown Panc-1 cells decreased in vitro cell migration, survival and proliferation, and reduced in vivo tumor growth. Two selected anti-AXL mAbs (D9 and E8), which inhibited phosphorylation of AXL and of its downstream target AKT without affecting growth arrest-specific factor 6 (GAS6) binding, induced downexpression of AXL by internalization, leading to an inhibition of proliferation and migration in the four pancreatic cancer cell lines studied. In vivo, treatment by anti-AXL mAbs significantly reduced growth of both subcutaneous and orthotopic pancreatic tumor xenografts independently of their KRAS mutation status. Our in vitro and preclinical in vivo data demonstrate that anti-human AXL mAbs could represent a new approach to the pancreatic cancer immunotherapy.

Conflict of interest statement

Conflict of interestWilhem Leconet, Christel Larbouret, Thierry Chardes, André Pèlegrin and Bruno Robert are inventors on anti-AXL mAb patents related to this work. The others authors declare that they have no competing interests.

Figures

Figure 1

AXL expression in pancreatic cancer cells. AXL protein was detected by IHC in 32 out of 42 PDAC (B – AXL-negative case; C,D,E – AXL-positive cases). Its strongest expression was observed in the invasive cells, localized at the periphery of the tumors and frequently of anaplastic morphology (C). In 2 cases AXL-expressing single invasive cells were observed (D; arrows). Two another cases had AXL-positive cells in the emboli (E; arrow).

Figure 2

AXL knockdown in the Panc-1 pancreatic cancer cell line affects cell viability, colony formation, migration and impairs tumor growth. Analysis of cell viability after five days in culture by MTS (A), colony formation after 14 days by Giemsa staining (B), cell migration after 24h of wound healing (C), and tumor growth (D) using Panc-1 sh-AXL1, sh-AXL2 or sh-CONTROL cell lines. ***p<0.001

Figure 3

The E8 and D9 anti-AXL mAbs are internalized, down-regulate AXL and inhibit the AKT signaling pathway and the MAPK pathway in a KRAS-wt cell line. (A) Internalization of the anti-AXL D9 and E8 mAbs in Panc-1 cells was detected by immunofluorescence. Cells were incubated or not with the anti-AXL mAbs at 4°C or 37°C for 1 hour and then with FITC-conjugated goat anti-mouse IgGs. (B) AXL expression in Capan-1, MiaPaCa-2, BxPC-3 and Panc-1 cells was analyzed by Western blotting at different time points after incubation with 100 μg/ml D9 and quantified in comparaison to GAPDH level. (C) Panc-1 and BxPC-3 cells were preincubated with 100 μg/ml D9 and E8 for 1.5h and then stimulated with 200 ng/ml rhGAS6 for 30 minutes. After cell lysis, phosphorylation of AXL (Y702), AKT (S473) and MAPK (T202, Y204) was analyzed by Western blotting. GAPDH served as loading control.

Figure 4

Anti-AXL mAbs inhibited migration, viability and induced ADCC. (A) Wound healing assay using Capan-1 cells: 24h post-injury, wound healing was evaluated in untreated cells and in cells stimulated or not with 200 ng/ml GAS6, following or not preincubation with 100 μg/ml E8 or D9 anti-AXL mAbs, as indicated. (B) Cell viability was assessed by the MTS assay after incubation with 25, 50 or 100 μg/ml of E8 or D9 anti-AXL mAbs for 5 days. Results are presented as the percentage of viability inhibition relative to the untreated (0% growth inhibition) and Triton-lysed cultures (100% growth inhibition). Each value represents the mean +/− SEM (n=6). (C) Concentration-dependent evaluation of anti-AXL-dependent ADCC activity against MiaPaCa-2-Luc cells by human PBMCs. MiaPaCa-2-Luc cells were first incubated with two concentrations of anti-AXL D9 or E8 or with positive control anti-HER2 trastuzumab (10 and 100 ng/mL) during 30 min at 37°C. Then the cells were incubated with hPBMC during 24 h (37°C) at an E:T ratio of 10:1. Viability of MiaPaCa-2-Luc cells was evaluated by measuring bioluminescence in the presence of luciferin. *p<0.05; **p<0.01; ***p<0.001.

Figure 5

Preclinical evaluation of the E8 and D9 anti-AXL mAbs in athymic mice subcutaneously xenografted with MiaPaCa-2 or BxPC-3 cells. (A) At day 30 (MiaPaCa-2 cells) and day 14 (BxPC-3 cells), the mice were treated with 15 mg/kg of E8 or D9 mAb, gemcitabine (150 mg/kg) or saline (n=10/group), twice per week for four weeks. Results are presented as the mean tumor volume ± SEM for each group. (B) Modified Kaplan-Meier survival curves represent the percentage of mice with tumor volume 3 as a function of time after-graft. (C) Benefit in days and percent of tumor free mice in both MiaPaCa-2 and BxPC-3 therapies summarized. (D) E8 or D9 mAb, but not saline (CTRL), induced AXL down-regulation in both tumor xenografts 7 days after the start of treatment. GAPDH served as loading control. (E) Immunohistochemistry analysis shows that cleaved caspase-3 expression is increased in MiaPaCa-2 tumor xenografts treated with D9 or E8, but not in saline-treated controls. **p<0.01; ***p<0.001

Figure 6

Effect of the anti-AXL mAb D9 in an orthotopic model of pancreatic cancer. MiaPaCa-2-Luc cells were injected in the pancreas of nude mice. (A) SPECT-CT imaging was carried out 24 and 48 h after injection of I125-labeled D9 (day 7 post-graft). (B) Tumor growth (n=10 per group) was evaluated by measuring the emitted luminescence once a week after luciferin injection. Bioluminescence intensity (p/s) is presented as a function treatment duration (days). (C) Representative bioluminescence imaging (following intraperitoneal injection of 0.1 mg/g Luciferin) of mice at the beginning (day 7) of the treatment with 15 mg/kg D9 twice a week for 4 weeks, and at day 35 post-graft. (D,E) At day 35, all mice were sacrificed and pancreatic tumors weighted and measured. **p<0,01; ***p<0.001