Afuco™ Anti-Human MCAM ADCC Recombinant Antibody (ABX-MA1), ADCC Enhanced (CAT#: AFC-496CL)

Figure 1 Western blot analysis of MUC18/MCAM in melanoma cell lines and HUVECs by ABX-MA1.

ABX-MA1 detected a single band of Mr 113,000 MUC18/MCAM protein on the metastatic (MUC18-positive) melanoma cell lines A375SM and WM2664 (Lanes 1 and 4). The Mr 113,000 MUC18 band was not detected in the MUC18-negative SB-2 cell line (Lane 2), but it was observed in SB-2 cells after transfection with the MUC18 gene (Lane 3). MUC18/MCAM was also detected on HUVECs (Lane 6), but not on nude mice endothelial cells (NME, Lane 5). The Mr 52,000 band corresponds to β-actin to show equal loading.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 2 FACS analysis for the expression of MUC18/MCAH on A375SM but not on SB-2 cells.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 3 Disruption of spheroid formation by ABX-MA1.

ABX-MA1 but not control IgG blocked spheroid formation in MUC18-expressing A375SM cells but not in MUC18-negative SB-2 cells.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 4 Effect of ABX-MA1 on tumor growth of human melanoma cells in nude mice.

A375SM (1 × 10⁵) or WM2664 (5 × 10⁵) cells were injected s.c. into nude mice (n = 10). Three days later, A375SM-bearing mice were treated with ABX-MA1 (1 mg, weekly, i.p.), whereas mice injected with WM2664 cells were treated with ABX-MA1 (100 μg, twice weekly, i.p.) or control IgG. The volume of the s.c. tumors was determined every 3–5 days. Treatment with ABX-MA1 significantly inhibited the growth of A375SM and WM2664 in nude mice.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 5 Effect of ABX-MA1 on proliferation of melanoma and HUVECs in vitro. A375SM, WM2664, and HUVECs were treated with 100 μg/ml ABX-MA1 or control IgG for 1–4 days.

Cell proliferation was determined by MTT assay. This is one of three representative experiments.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 6 Effect of ABX-MA1 on MMP-2

A, down-regulation of MMP-2 activity in melanoma cells by ABX-MA1. A375SM melanoma cells were treated with ABX-MA1 (100 μg/ml) or control IgG for 5 days and analyzed for MMP-2 activity by zymography. Note that ABX-MA1 but not control IgG suppressed MMP-2 activity in melanoma cells. B, effect of ABX-MA1 on MMP-2 promoter. Melanoma cells were treated with 100 μg/ml ABX-MA1, control IgG, or CMEM for 4 days, and then PGL3-MMP-2 reporter plasmid was cotransfected with pB-Actin-RL reporter gene into the melanoma cells. Fold decrease in luciferase activity was calculated relative to the luciferase activity in untreated cells, which was assigned the value of 1.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 7 Effect of ABX-MA1 on MMP-2 expression in vivo.

MMP-2 expression was determined by IHC in specimens derived from A375SM and WM2664 implanted tumors. MMP-2 expression was reduced in tumors taken from ABX-MA1-treated mice.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 8 Tumor MVD and apoptosis (TUNEL) in s.c. melanoma xenografts.

A375SM and WM2664 cells were injected s.c. into nude mice and treated with ABX-MA1 or control IgG. Thirty to 60 days later, the resulting s.c. tumors with similar size were resected and processed for immunohistochemical analysis (CD31 and TUNEL staining).

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 9 Effect of ABX-MA1 on attachment of melanoma cells to HUVECs.

A, A375SM (MUC18-positive) and SB-2 (MUC-18-negative) cells were cocultured with HUVECs in the presence of ABX-MA1 or control IgG. Whereas very few SB-2 cells attached to HUVECs, A375SM did attach to HUVECs, and this interaction was inhibited by ABX-MA1 but not by control IgG. B, quantitation of the interaction of A375SM with HUVECs is summarized by a bar graph and demonstrates that ABX-MA1 inhibited A375SM-HUVEC interaction by 80%.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.

Figure 10 Effect of ABX-MA1 on vessel-like tube formation by HUVECs.

A, untreated HUVECs formed the tube-like vessels. B, treatment with control IgG did not disrupt the vessel-like tube formation. C, ABX-MA1 pretreatment disrupted vessel-like tube formation by HUVECs. D, no effect was observed when ABX-MA1 was added after the network had been established.

Mills, L., Tellez, C., Huang, S., Baker, C., McCarty, M., Green, L., ... & Bar-Eli, M. (2002). Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer research, 62(17), 5106-5114.