Mouse Anti-GD2 Recombinant Antibody (clone 14G2a) (CAT#: PABL-100)

Figure 1 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 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. 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.

Alvarez-Rueda, N., Desselle, A., Cochonneau, D., Chaumette, T., Clemenceau, B., Leprieur, S.,... & Saba, J. (2011). A monoclonal antibody to O-acetyl-GD2 ganglioside and not to GD2 shows potent anti-tumor activity without peripheral nervous system cross-reactivity. PloS one, 6(9), e25220.

Figure 2 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). Empty columns, irrelevant antibody; black columns, mAb 8B6; grey columns, mAb 14G2a.

Alvarez-Rueda, N., Desselle, A., Cochonneau, D., Chaumette, T., Clemenceau, B., Leprieur, S.,... & Saba, J. (2011). A monoclonal antibody to O-acetyl-GD2 ganglioside and not to GD2 shows potent anti-tumor activity without peripheral nervous system cross-reactivity. PloS one, 6(9), e25220.

Figure 3 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). (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).

Alvarez-Rueda, N., Desselle, A., Cochonneau, D., Chaumette, T., Clemenceau, B., Leprieur, S.,... & Saba, J. (2011). A monoclonal antibody to O-acetyl-GD2 ganglioside and not to GD2 shows potent anti-tumor activity without peripheral nervous system cross-reactivity. PloS one, 6(9), e25220.

Figure 4 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 (□).

Alvarez-Rueda, N., Desselle, A., Cochonneau, D., Chaumette, T., Clemenceau, B., Leprieur, S.,... & Saba, J. (2011). A monoclonal antibody to O-acetyl-GD2 ganglioside and not to GD2 shows potent anti-tumor activity without peripheral nervous system cross-reactivity. PloS one, 6(9), e25220.

Figure 5 Expression of GD2 on the cell surface of EL-4, IMR-32, and mS tumor cell lines.

Flow cytometry analysis of the cells stained with anti-GD2 antibodies conjugated with AlexaFluor488 (14G2a antibodies; 5 μg/ml; see Methods) is shown in (A). Filled histograms (red color) show staining with anti-GD2 mAbs, empty histograms – staining with an isotype control. Confocal imaging of EL-4, IMR-32, and mS cells stained with anti-GD2 conjugated with AlexaFluor488 (14G2a antibodies; 5 μg/ml; see Methods) is shown in (B). The staining with anti-GD2 mAb is shown in green color; the nuclei were counterstained with Hoechst 33342 (shown in blue). Bar scale: 50 μm.

Doronin, I. I., Vishnyakova, P. A., Kholodenko, I. V., Ponomarev, E. D., Ryazantsev, D. Y., Molotkovskaya, I. M., & Kholodenko, R. V. (2014). Ganglioside GD2 in reception and transduction of cell death signal in tumor cells. BMC cancer,14(1), 295.

Figure 6 The cytotoxic effects of two types of anti-GD2 antibodies on GD2-positive tumor cell lines.

Phase-contrast images of GD2-positive tumor cell lines EL-4, IMR-32, and mS after 24 h of incubation with or without anti-GD2 mAbs, 14G2a (5 μg/ml) and ME361 (5 μg/ml) are shown in (A). In (A), bar scale: 50 μm. Analysis of DNA fragmentation (PI assay; see Methods) of GD2-positive tumor cells EL-4, IMR-32, mS treated with GD2 mAbs 14G2a (5 μg/ml) and ME361 (5 μg/ml) is shown in (B). In (B), the percentages of the cells with fragmented DNA in hypodiploid peaks are shown for each histogram.

Doronin, I. I., Vishnyakova, P. A., Kholodenko, I. V., Ponomarev, E. D., Ryazantsev, D. Y., Molotkovskaya, I. M., & Kholodenko, R. V. (2014). Ganglioside GD2 in reception and transduction of cell death signal in tumor cells. BMC cancer,14(1), 295.

Figure 7 Comparison of the influence of anti-GD2 antibodies on viability of GD2-positive vs. GD2-negative tumor cell lines.

The viability of GD2-positive (EL-4, IMR-32, mS) and GD2-negative (Neuro-2A, A375, Jurkat) tumor cells was assessed for the cells incubated with various concentration of anti-GD2 mAbs for 72 h using MTT assay as described in Methods. Results are shown for two monoclonal anti-GD2 antibodies 14G2a (A) and ME361 (B). Mean ± S.E. of three separate experiments is shown, statistical analysis was performed using two-way analysis of variance method for concentrations of 0.31 – 10 μg/ml (A), and for concentrations 2.5 – 10 μg/ml (B). The differences between GD2-positive and GD2-negative groups were statistically significant (***, P < 0.001) as determined by Student-Newman-Keuls post-hoc analysis.

Doronin, I. I., Vishnyakova, P. A., Kholodenko, I. V., Ponomarev, E. D., Ryazantsev, D. Y., Molotkovskaya, I. M., & Kholodenko, R. V. (2014). Ganglioside GD2 in reception and transduction of cell death signal in tumor cells. BMC cancer,14(1), 295.

Figure 8. Analysis of apoptotic volume decrease and the loss of plasma membrane integrity for EL-4 lymphoma cells treated with anti-GD2 antibodies.

Apoptotic volume decrease (AVD) (A) and cell membrane permeability (B) were analyzed for the control (untreated) EL-4 cells or after 2 h of incubation with anti-GD2 mAbs 14G2a (5 μg/ml), or Staurosporine (500 nM) that was used as positive control for induction of apoptosis (see Methods). In (A), R1 – region of viable cells, R2 – region of cells with AVD, and R3 – region of cell debris. In (B), percentages of 7-AAD positive cells are shown for each histogram.

Doronin, I. I., Vishnyakova, P. A., Kholodenko, I. V., Ponomarev, E. D., Ryazantsev, D. Y., Molotkovskaya, I. M., & Kholodenko, R. V. (2014). Ganglioside GD2 in reception and transduction of cell death signal in tumor cells. BMC cancer,14(1), 295.