MOC31PE immunotoxin - targeting peritoneal metastasis from epithelial ovarian cancer

Abstract

Peritoneal metastasis (PM) is an important feature of epithelial ovarian cancer (EOC) and is a frequent site of drug resistant disease recurrence, identifying PM-EOC an important clinical challenge. The MOC31PE immunotoxin targets and kills tumor cells expressing the epithelial cell adhesion molecule (EpCAM), which is highly expressed in EOC, and MOC31PE is being investigated for use in treatment of PM-EOC. The efficacy of MOC31PE treatment alone and in combination with cytotoxic drugs was investigated in two human EpCAM expressing EOC cell lines, B76 and MDHA-2774, in vitro and in corresponding mouse models mimicking PM-EOC. MOC31PE efficaciously killed tumor cells alone and showed equal or superior activity in vitro (paclitaxel, cisplatin, carboplatin) and in vivo (paclitaxel, mitomycin C) compared to the investigated cytotoxic drugs. Additive, or importantly, no antagonistic effects were observed in combination experiments. In ex vivo cell culture, the cytotoxic effect of MOC31PE was studied on freshly isolated surgical EOC samples. All investigated fresh EOC samples expressed EpCAM and MOC31PE effectively reduced cell viability in ex vivo cultures. In conclusion, these results, together with our previous preclinical and clinical experience, support development of MOC31PE for treatment of PM-EOC in combination with currently used cytotoxic drugs.

Keywords: EpCAM; MOC31PE immunotoxin; animal models; chemotherapy; peritoneal metastasis of epithelial ovarian cancer.

Figure 1. MOC31PE in combination with paclitaxel causes additive cytotoxicity in vitro

(A) B76 and 2774 cells were incubated with paclitaxel (50 nM) and/or carboplatin (100 µM) and/or cisplatin (33 µM) and/or MOC31PE (10 ng/ml) for 24 h. Cell viability is expressed as a percentage (mean {plus minus} SD) of the value obtained in vehicle treated cells. The assays were performed in triplicate, and repeated at least three times. **; p<0.05 compared to vehicle treated cells, *; p<0.05 compared to monotherapies. (B) Western immunoblots of cell lysates from B76 and 2774 cells treated for 24 h with MOC31PE (10 ng/ml) and/or paclitaxel (50 nM) and/or carboplatin (100 µM). The 116- and 85-kDa bands represent the uncleaved and cleaved versions of PARP, respectively.

Figure 2. Characterization of peritoneal metastasis models of human epithelial ovarian carcinoma

(A) Photographs taken at autopsy of a representative mouse on day 24 after i.p. injection of 2.5x10⁶ B76 cells. A mean number of 54 (SD 17) tumor nodules were seen distributed all over the peritoneal surfaces, including ovaries, kidneys, bladder, intestine and spleen. (B) Representative images of B76 tumor sections after hamatoxylin-eosin (H&E) and immunohistochemical staining. H&E stained B76 tumor section (top), strong EpCAM expression in the plasma membrane (middle), and both cytoplasmic and nuclear Pax8 staining (lower) picture. Magnification x40. (C) Photographs taken at autopsy of a mouse on day 23 after i.p. injection of 2.5x10⁶ 2774 cells. A mean number of 25 (SD 12) tumor nodules with a distribution similar to that seen with B76 cells. EpCAM-positive cancer cells in the ascites fluid were detected and isolated with immunomagnetic MOC31-coated beads (two lower pictures). (D) Immunoblots showing EpCAM expression (lane 1 and 2) in isolated 2774 cells from ascites of two representative animals with anti-α-tubulin antibody as loading control.

Figure 3. MOC31PE increases survival of mice with B76 and 2774 peritoneal metastases

Kaplan-Meier survival curves of mice injected i.p. with (A) B76 and (B) 2774 cells. B76 animals were treated i.p. 24 h later with one dose of MOC31PE (150 µg/kg) or vehicle whereas 2774 mice received a single treatment dose either day 1, day 7 or day 11 after injection of the tumor cells. The mean survival time (MST) is summarized in the tables under the graphs.

Figure 4. In vivo efficacy of MOC31PE, paclitaxel and mitomycin C as single agents and in combination experiments

Kaplan-Meier survival curves of groups of at least five mice injected with B76 (A) or 2774 (B) cells and treated i.p. 24 h later with single doses of MOC31PE and paclitaxel (7.5 mg/kg) alone and in combination, with vehicle as control. The MOC31PE dose was low in B76 mice (50 µg/kg) and high in 2774 mice (150 µg/kg). The mean survival times (MST) and p-values are summarized in the tables under the graphs. (C) Kaplan-Meier survival curves of groups of at least five mice inoculated with B76 cells and treated i.p. 24 h later with single doses of a low MOC31PE (32 µg/kg) and a low mitomycin C (1mg/kg) or the combination, and with vehicle as a control. The MST summary for the different groups is shown in the table under the graph. The significance between treatment and vehicle animals were determined by the log-rank test.

Figure 5. Ex vivo effects of MOC31PE on ovarian cancer cells freshly isolated from patients’ peritoneal metastases

The picture shows EpCAM-positive cells isolated from disaggregated tumor tissue from patient 1 using immunomagnetic MOC31-coated beads. Cells from patient 1 were treated with MOC31PE (0.1-10 ng/ml) or vehicle and incubated at 37°C for 24 h, demonstrating a dose-response relationship with less than 40% cell viability at 10 ng/ml. Disaggregated tumor tissue from patient number 2, 3 and 4 was treated with MOC31PE (10 ng/ml), with cell viability of 60 and 75% compared to vehicle (p<0.05). The assays were performed in at least six parallels