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. Author manuscript; available in PMC: 2018 Aug 18.
Published in final edited form as: ACS Chem Biol. 2017 Jun 28;12(8):2085–2096. doi: 10.1021/acschembio.7b00305

Figure 5. Glycan-MUC16 ectodomain antibody function.

Figure 5

A) Anti-gylcosylation site antibodies block MUC16-enhanced matrigel invasion. Each of the four anti–glycan-MUC16-ectodomain antibodies inhibit the invasion of three different MUC16-positive ovarian cancer cell lines. Results are expressed as percentages compared to the untreated control. All of the inhibitory effects were significant compared to untreated control (p<0.001).

B) Immunoprecipitation (IP) of EGFR, MUC16c57-114-pFUSE, and Galectin-3 is blocked by anti glycosylation site antibodies. The presence of an anti-MUC16 N-glycosylation antibody (18C6) (lanes 9-11) completely eliminates interactions between MUC16–Galectin-3 and EGFR (lanes 5-8). The 18C6 antibody (lanes 9-11) also blocks interaction between MUC16, Galectin-3 and Integrin β1 proteins (lanes 5–8).

C) Anti-glycosylation site antibody 18C6 blocks MUC16-induced oncogene expression. As in Figure 1D, the pAKT, pERK1/2, pSRC, and pEGF receptor (pEGFR) signaling pathways were examined, indicating increased phosphorylation of AKT (S473), ERK1/2 (pT202/Y204), pSRC(y416), and pEGFR(y1068) in A2780-MUC16c114 and A2780-MUC16c344 cells compared to the phr control cells. Eighteen-hour exposure of 18C6 antibody to A2780-MUC16c114 and A2780-MUC16c344 cells blocks MUC16c114 and MUC16c344 oncogene activation in A2780 cells.

D) Anti-glycosylation site antibody blocks SKOV3-MUC16c344 and A2780-MUC16c344 tumor growth in athymic female nude mice. SKOV3-MUC16c344 or A2780-MUC16c344 tumor cells were each introduced into the flank of 20 nu/nu mice. Ten mice were treated intravenously from day 0 with purified 10C6 (Panel 5Di) or 18C6 (Panel 5Dii) GlcNAc2–MUC16 monoclonal antibody at 100 μg/mouse twice per week for 4 weeks. Tumors were measured by calipers twice/week. The differences in mean tumor volume were significantly decreased (p=0.0004) with 10C6 monoclonal antibody-treated mice bearing SKOV3-MUC16c344. The mice bearing A2780-MUC16c344 tumors were treated twice per week with the 18C6 antibody. The mean tumor volume was significantly decreased (p=0.02) with 18C6 monoclonal antibody-treated mice bearing A2780-MUC16c344 tumors compared to untreated A2780-MUC16c344 tumors. The inserts in the figure show the matrigel invasion assay with the same cell lines performed in the presence and absence of purified 10C6 or 18C6 monoclonal antibody.