Therapeutic activity of glycoengineered anti-GM2 antibodies against malignant pleural mesothelioma

Abstract

Malignant pleural mesothelioma (MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action. Ganglioside GM2 is a glycolipid that has been shown to be overexpressed in various types of cancer. However, there are no published reports regarding the use of GM2 as a potential therapeutic target in cases of MPM. In this study, we evaluated the efficacy of the anti-GM2 antibody BIW-8962 as an anti-MPM therapeutic using in vitro and in vivo assays. Consequently, the GM2 expression in the MPM cell lines was confirmed using flow cytometry. In addition, eight of 11 cell lines were GM2-positive (73%), although the GM2 expression was variable. BIW-8962 showed a significant antibody-dependent cellular cytotoxicity activity against the GM2-expressing MPM cell line MSTO-211H, the effect of which depended on the antibody concentration and effector/target ratio. In an in vivo orthotropic mouse model using MSTO-211H cells, BIW-8962 significantly decreased the incidence and size of tumors. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. Fifty-eight percent of the MPM tumors were positive for GM2, with individual variation in the intensity and frequency of staining. These data suggest that anti-GM2 antibodies may become a therapeutic option for MPM patients.

Keywords: Antibodies; antibody-dependent cell cytotoxicity; ganglioside GM2; mesothelioma; therapeutics.

Figure 1

Expression levels of ganglioside GM2 were evaluated in malignant pleural mesothelioma cell lines. Cells were detached and incubated with BIW-8962 or anti-dinitrophenol (DNP) antibodies on ice for 30 min. Bound Abs were detected with FITC-conjugated goat anti-human IgG Abs. The fluorescence intensity of the stained cells was measured using flow cytometry, and the mean fluorescence intensity was calculated. The open red histograms represent BIW-8962-stained samples and the filled blue histograms represent anti-DNP antibody-stained samples. The relative fluorescent intensity (RFI) versus anti-DNP is indicated.

Figure 2

Anti-GM2 antibody BIW-8962 exerted antibody-dependent cellular cytotoxicity activity against MSTO-211H malignant pleural mesothelioma cells. Human peripheral blood mononuclear cells were purified from healthy donors and used as effector cells. MSTO-211H cells (target cells) were incubated with effector cells (effector/target = 25/1, 50/1, and 100/1) and antibodies (BIW-8962 or anti-dinitrophenol antibodies) at 37°C for 4 h. The released lactate dehydrogenase activity was measured and the % cytotoxicity was calculated. The experiments were carried out in triplicate, and the values are expressed as the mean of the values for four donors ± SD. *P < 0.01, **P < 0.001 BIW-8962 treatment versus anti-dinitrophenol antibody treatment.

Figure 3

Anti-GM2 antibody BIW-8962 showed therapeutic activity in an MSTO-211H orthotropic mouse model. MSTO-211H malignant pleural mesothelioma cells were inoculated into the thoracic cavity in SCID mice, and the animals were treated with BIW-8962 and/or human peripheral blood mononuclear cells (MNC). The mice were then i.v. administered BIW-8962 and/or MNC on days 7 and 14. At 3 weeks after tumor cell inoculation, the mice were sacrificed and their tumor weights were measured. The bars represent the mean of the group data. *P < 0.05 and **P < 0.01 between each treatment and the control groups.

Figure 4

Ganglioside GM2 expression in clinical malignant pleural mesothelioma specimens. Frozen sections obtained from 26 mesothelioma patients were incubated in DAPI at room temperature for 20 min. After washing, the sections were incubated in 5% FBS–PBS for 10 min for blocking and subsequently incubated in 30 μg/mL BIW-8962 or anti-dinitrophenol antibodies conjugated with Alexa Fluor488 at 4°C for 6 h. Representative images are shown. Samples of patients #12 (a), 19 (b), 20 (c), and 23 (d) are shown.