Human Anti-EGFR Recombinant Antibody (clone Cetuximab (IMC-C225)) (CAT#: PABW-039)

Figure 1 Immunoblot of EGFR and tyrosine-phosphorylated EGFR from pancreatic carcinoma cells, BxPC-3, and MiaPaCa-2.

The EGFR-overexpressing human epidermoid cell line A431 was used as a positive control. Total cellular lysates were collected from cells treated for 24 h with IMC-C225, then exposed to EGF for 5 min. Immunoblot analysis for EGFR expression showed a low but detectable level of receptor associated with the pancreatic cell lines. IMC-C225 treatment did not have any effect on EGFR protein levels; however, the antibody did block ligand binding as detected by the dramatic reduction in EGF-induced tyrosine phosphorylation of EGFR. Actin protein was used as a control for protein loading.

Buchsbaum, D. J., Bonner, J. A., Grizzle, W. E., Stackhouse, M. A., Carpenter, M., Hicklin, D. J., ... & Raisch, K. P. (2002). Treatment of pancreatic cancer xenografts with Erbitux (IMC-C225) anti-EGFR antibody, gemcitabine, and radiation. International Journal of Radiation Oncology* Biology* Physics, 54(4), 1180-1193.

Figure 2 In vitro inhibition of pancreatic carcinoma cell growth.

BxPC-3 and MiaPaCa-2 cells were treated with IMC-C225 (C225) on Day 0, gemcitabine (Gem) on Day 1 for 24 h, followed by 60Co irradiation (RT) on Day 2, or the combination of each agent, then counted on Day 4. BxPC-3 cells treated with the combination of all three agents were significantly inhibited in cell proliferation as compared to the untreated control cells (p<0.05). Similar results were observed with the MiaPaCa-2 cells, with the three-agent combination showing a significant inhibition of cell proliferation as compared to the untreated and IMC-C225 treatment groups. Data points are the average SEM of three independent experiments, each done in quadruplicate cultures, then normalized to the untreated control (100%).

Buchsbaum, D. J., Bonner, J. A., Grizzle, W. E., Stackhouse, M. A., Carpenter, M., Hicklin, D. J., ... & Raisch, K. P. (2002). Treatment of pancreatic cancer xenografts with Erbitux (IMC-C225) anti-EGFR antibody, gemcitabine, and radiation. International Journal of Radiation Oncology* Biology* Physics, 54(4), 1180-1193.

Figure 3 Analysis of pancreatic carcinoma cells for the induction of early apoptosis. Floating and adherent cells were collected 4 days posttreatment, stained with annexin V-FITC and propidium iodide, then analyzed by FACS using CellQuest software.

A significant increase in apoptosis was observed with the BxPC-3 cells after treatment with the three-agent combination as compared to the untreated, radiation (RT), IMC-C225, gemcitabine (Gem), and radiation C225 treatment groups (p<0.001). The BxPC-3 cells showed a significant increase in apoptotic cell death after treatment with Gem C225 and Gem radiation as compared to the untreated and radiation treatment groups (p <0.001). With MiaPaCa-2 cells, the three-agent combination and the two-agent combination of Gem radiation showed a significant increase in apoptotic cells as compared to all other treatment groups (p<0.001). Also the MiaPaCa-2 cells treated with Gem or Gem C225 showed a significant increase in apoptosis as compared to untreated, C225, radiation, and C225 radiation treatment groups (p<0.001). Data points are the average SEM of three independent experiments done in triplicate (n=9).

Buchsbaum, D. J., Bonner, J. A., Grizzle, W. E., Stackhouse, M. A., Carpenter, M., Hicklin, D. J., ... & Raisch, K. P. (2002). Treatment of pancreatic cancer xenografts with Erbitux (IMC-C225) anti-EGFR antibody, gemcitabine, and radiation. International Journal of Radiation Oncology* Biology* Physics, 54(4), 1180-1193.

Figure 4 TUNEL result of IMC-C225.

(A) An apparently viable area of a MiaPaCa-2 tumor xenograft from an untreated nude mouse; about 5%–10% of cells demonstrate apoptosis identified by the TUNEL technique. (B) A viable area of a MiaPaCa-2 xenograft tumor from a mouse treated for 1 week with IMC-C225 (1 mg every 3 days 2), gemcitabine (120 mg/kg 1 day after the first dose of mAb), and radiation (3 Gy at 1 day after gemcitabine); at 4 days after radiation, the mice were killed. About 30%–40% of cells exhibit apoptosis that is higher compared to the basal level in untreated tumors. (C) Untreated mouse with a MiaPaCa-2 tumor xenograft was injected i.p. with BrdU and killed 2 h later; about 70%–80% of the cells were
undergoing proliferation. (D) A marked reduction in proliferation as detected by BrdU staining in treated MiaPaCa-2 tumors (10%–15%) compared to the basal level in untreated tumors (60%–70%).

Buchsbaum, D. J., Bonner, J. A., Grizzle, W. E., Stackhouse, M. A., Carpenter, M., Hicklin, D. J., ... & Raisch, K. P. (2002). Treatment of pancreatic cancer xenografts with Erbitux (IMC-C225) anti-EGFR antibody, gemcitabine, and radiation. International Journal of Radiation Oncology* Biology* Physics, 54(4), 1180-1193.