A monoclonal antibody to O-acetyl-GD2 ganglioside and not to GD2 shows potent anti-tumor activity without peripheral nervous system cross-reactivity

Abstract

Background: Monoclonal antibodies (mAb) against GD2 ganglioside have been shown to be effective for the treatment of neuroblastoma. Beneficial actions are, however, associated with generalized pain due to the binding of anti- GD2 mAbs to peripheral nerve fibers followed by complement activation. Neuroblastoma cells that express GD2 also express its O-acetyl derivative, O-acetyl- GD2 ganglioside (OAcGD2). Hence, we investigated the distribution of OAcGD2 in human tissues using mAb 8B6 to study the cross-reactivity of mAb 8B6 with human tissues.

Methodology/principal findings: The distribution of OAcGD2 was performed in normal and malignant tissues using an immunoperoxydase technique. Anti-tumor properties of mAb 8B6 were studied in vitro and in vivo in a transplanted tumor model in mice. We found that OAcGD2 is not expressed by peripheral nerve fibers. Furthermore, we demonstrated that mAb 8B6 was very effective in the in vitro and in vivo suppression of the growth of tumor cells. Importantly, mAb 8B6 anti-tumor efficacy was comparable to that of mAb 14G2a specific to GD2.

Conclusion/significance: Development of therapeutic antibodies specific to OAcGD2 may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of antibodies.

Figure 1. Expression of OAcGD2 and GD2 ganglioside antigens on peripheral nerves and neuroblastomas.

An immunoperoxydase assay was performed as described in Material and Methods. Strong immunostaining was detected on neuroblastoma cells (A) with either mAb 8B6 (2) or mAb 14G2a (3). No staining was observed on peripheral nerves (B) with mAb 8B6 specific for OAcGD2 (2). Myelin sheaths in the peripheral nerves (B) were strongly stained with mAb 14G2a against GD2 (3). The mouse IgG3 mAb negative control from AbD serotec (Oxford, UK) was used as a negative control (1). Scale bar  = 50 µm.

Figure 2. Antibody 8B6 and mAb 14G2a each induce viability inhibition and apoptosis of EL4 cells.

(A), EL4 cell line was treated for 24 hours with various concentrations of mAb 8B6 (), mAb 14G2a (▪) and a control 4F6 antibody (▴). Viability was assessed as described in « Materials and Methods » by adding the methylthiazole tetrazolium salt during 4 hours (MTT assay). Optical density was recorded at 570 nm. The data are presented as the mean ± SD for three independent experiments, each in triplicate. (B), On the treated right column, EL4 cells were exposed to either 50 µg/mL of mAb 8B6 or mAb 14G2a for 24 hours and then double stained with fluorescein-isothiocyanate-conjugated F(ab')₂ fragments of goat anti-mouse IgG (H+L). After permeabilization, cells were stained with Apo2.7-PE antibody as described in ‘ Materials and Methods’. The percentage of double-positive cell in the untreated EL4 tumor cells is indicated in the left column. Numbers in quadrants represent the percentage of cells in each section of the quadrant.

Figure 3. Activation of complement by mAb 8B6 and mAb 14G2a.

Complement-dependent specific lysis was determined for the EL4 cell line (A), the NXS2 cell line (B), and the OAcGD2/GD2-negative Neuro 2A cells (C) as described in Material and Methods. Empty columns, irrelevant antibody; black columns, mAb 8B6; grey columns, mAb 14G2a.

Figure 4. ADCC of mAb 8B6 and mAb 14G2a.

(A) The A-LAK killer activity with mAb 8B6 and mAb 14G2a with EL4 target cells at the E/T ratio 12 to 1 (empty columns), 25 to 1 (grey columns), and at the E/T ratio 50 to 1 (black columns) as described in Material and Methods. (B) The A-LAK killer efficiency in the ADCC assays was tested using the sensitive YAC-1 target cells. (C) ADCC activity with mAb 8B6 and mAb 14G2a against the OAcGD2/GD2-negative Neuro 2A cells used as a negative control, at the E/T ratio 12 to 1 (empty columns), 25 to 1 (grey columns), and at the E/T ratio 50 to 1 (black columns). (D) ADCC mediated by mAb 8B6 (grey column) and mAb 14G2a (black column) with EL4 target cells at varying antibody concentrations. The results were compared to the effect of equal amount of irrelevant antibody used as a negative control (n = 3).

Figure 5. Survival of C57BL/6 mice inoculated with EL4 cells treated with either 8B6 or 14G2a mAb.

Mice (n = 10) were inoculated with 10⁴ EL4 cells subcutaneously and then treated with either PBS or 70 µg of each antibody, twice weekly for 3 weeks. PBS (), mAb 8B6 (○), mAb 14G2a (▿), control 4F6 antibody (□).