High efficacy of CpG-ODN, cetuximab and cisplatin combination for very advanced ovarian xenograft tumors

Abstract

Background: To mimic clinical treatment situations in advanced human ovarian disease, we tested the efficacy of CpG-oligodeoxynucleotides (CpG-ODN), synthetic DNA sequences recognized by Toll-like receptor 9 and able to induce innate/adaptive immune responses, in combination with other possible therapeutic reagents in ovarian carcinoma ascites-bearing athymic mice.

Methods: Mice injected i.p. with IGROV-1 ovarian cancer cells were treated at different stages of ascites progression for 4 weeks with CpG-ODN, alone or in combination with Bevacizumab, Polyinosinic:Polycytidylic acid (Poly(I):Poly(C)), Gefitinib, Cetuximab and Cisplatin. Median survival time (MST) was calculated for each group. IGROV-1 cells treated or not with Cetuximab were assayed for antibody-dependent cellular cytotoxicity by 51Cr-release assay, and for macrophage antibody-dependent cell-mediated phagocytosis by flow cytometry.

Results: In mice treated when ascitic fluid began to accumulate, CpG-ODN combined with Bevacizumab, Poly(I):Poly(C) or Gefitinib did not significantly increase MST as compared with that using CpG-ODN alone, whereas MST in mice treated with CpG-ODN plus Cetuximab was significantly increased (>103 days for combination vs 62 days for CpG alone; P = 0.0008), with 4/8 mice alive at the end of the experiment. In experiments in mice showing increased abdominal volume and body weight (27.9 ± 0.8 g after vs 23 ± 1.1 g before tumor cell injection), treatment with Cisplatin in addition to CpG-ODN/Cetuximab led to significantly increased MST (105.5 days; P = 0.001), with all mice still alive at 85 days, over that using CpG-ODN/Cetuximab (66 days), Cetuximab/Cisplatin (18.5 days), Cisplatin (23 days) or saline (16 days). At a very advanced stage of disease (body weight: 31.4 ± 0.9 g), when more than half of control mice had to be sacrificed 6 days after starting treatments, the triple-combination therapy still increased MST (45 days; P = 0.0089) vs controls.

Conclusions: CpG-ODN combination therapies that enhance the immune response in the tumor microenvironment and concomitantly target tumor cells are highly efficacious even in experimental advanced malignancies. Although differences in the distribution of TLR9 in mice and humans and the enrichment of this receptor on innate immune cells of athymic mice must be considered, our results indicate a promising strategy to treat ovarian cancer patients with bulky ascites.

Figure 1

EGFR expression in IGROV-1 cells. Expression of EGFR in IGROV-1 cells evaluated by FACS. Thin and bold lines represent Alexa Fluor 488-secondary antibody and Cetuximab, respectively.

Figure 2

Kaplan-Meier plot of percent survival over time among IGROV-1 ovarian tumor-bearing mice. At 7 days after tumor cell injection, mice were treated i.p. with CpG-ODN (20 μg/mouse, 5 days/week for 4 weeks) in combination with: Poly(I):Poly(C) (20 μg/mouse at 2- to 3-day intervals); Bevacizumab (5 mg/Kg at 3- to 4-day intervals); Gefitinib (100 mg/mouse, 5 days/week) or Cetuximab (1 mg/mouse at 3- to 4-day intervals). Single agents were also tested. Control mice received saline. N = number mice/group.

Figure 3

Effect of Cetuximab pretreatment on phagocytosis of IGROV-1 cells. IGROV-1 target cells were stained green with PKH67 (A, right lower quadrant) and RAW264.7 effector cells were stained red with PKH26 (B, left upper quadrant). Tumor targets were pre-incubated for 72 h with 5 μg/ml Cetuximab (F,G,H) or left untreated (C,D,E). At the end of treatment, target and effector cells were mixed at effector/target (E/T) ratio of 3:1 in complete medium and incubated for an additional 12 h in overload conditions of monoclonal antibody (10 μg/ml). The percentage of double-positive cells present in the upper right quadrant (quadrant Q2) of the dot plots represents the percentage of RAW264.7 cells phagocytosing green-stained tumor cells. Data were obtained in triplicate and are representative of one of three experiments with similar results.

Figure 4

Kaplan-Meier plot of percent survival over time in advanced-stage IGROV-1 ovarian tumor-bearing mice. (A) Mice selected for the presence of evident and established ascites from a large group of mice injected i.p. 11 days before with IGROV-1 cells (mean body weight ± SEM 27.89 ± 0.84 g vs 23.00 ± 1.08 g before tumor cell injection) were treated with saline, Cisplatin (3 mg/kg, once per week), CpG-ODN (20 μg/mouse, 5 days/week for 4 weeks) plus Cetuximab (1 mg/mouse at 3- to 4-day intervals), CpG-ODN plus Cisplatin, Cetuximab plus Cisplatin, and CpG-ODN plus Cetuximab and Cisplatin. (B) Mice selected for more advanced-stage disease (mean body weight ± SEM 31.4 ± 0.9 g vs 24.89 ± 0.68 g before tumor cell injection) were treated with saline or CpG-ODN plus Cetuximab and Cisplatin. N = number mice/group.