Anti-Human CD3E Recombinant Antibody (Ertumaxomab) (CAT#: TAB-H28)

Figure 1 HER2/neu-expressing profiles of used cell lines: A, SK-BR-3, (B) HCT-8, (C) BT-20, and (D) SK-LU-1.

FACS, fluorescence activated cell sorter histogram, FL1-H = 2502A + secondary detection antibody rat anti-mouse IgG H+L FITC, mouse IgG2a isotype control Me361 (TRION Research) + secondary detection antibody rat anti-mouse IgG H+L FITC; MFI, mean fluorescence intensity; SABC, specific antigen binding capacity as determined by DAKO QIFIKIT

Jäger, M., Schoberth, A., Ruf, P., Hess, J., & Lindhofer, H. (2009). The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2. Cancer research, 69(10), 4270-4276.

Figure 2 Cytotoxicity against SK-BR-3 cells (HER2 high, HER2 ampl+) at E/T ratios of (A) 20:1 and (B) 5:1 mediated by trastuzumab (5,000–0.001 ng/mL) or ertumaxomab (100–0.001 ng/mL).

Cytotoxicity experiments were performed thrice and samples measured in duplicates with 2 × 105 PBMC of 3 different healthy donors; sample PBMC + SK-BR-3 + antibody; allogeneic reaction, PBMC + SK-BR-3 cells; points, mean; bars, SD.

Jäger, M., Schoberth, A., Ruf, P., Hess, J., & Lindhofer, H. (2009). The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2. Cancer research, 69(10), 4270-4276.

Figure 3 Cytotoxicity of ertumaxomab and trastuzumab against tumor cells with a low HER2/neu expression profile.

Cytotoxicity against (A) HCT-8 cells (HER2 low, HER2 ampl−; E/T, 20:1); (B) HCT-8 cells (E/T, 7:1); (C) BT-20 cells (HER2 low, HER2 ampl−; E/T, 20:1) and (D) SK-LU-1 cells (HER2 low, HER2 ampl−; E/T, 20:1) mediated by trastuzumab (5,000–0.001 ng/mL) or ertumaxomab (100–0.001 ng/mL). Cytotoxicity experiments were performed thrice and samples measured in duplicates with 2 × 105 PBMC of 3 different healthy donors; sample: PBMC + HCT-8 + antibody; allogeneic reaction: PBMC + HCT-8 cells. Points, mean; bars, SD.

Jäger, M., Schoberth, A., Ruf, P., Hess, J., & Lindhofer, H. (2009). The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2. Cancer research, 69(10), 4270-4276.

Figure 4 Cytokines induced by ertumaxomab in the presence of HCT-8 target cells and PBMC.

A-C, influence of ertumaxomab-induced cytokines IFN-γ, IL-6, and IL-2 on freshly plated HCT-8 cells (HER2 low, HER2 ampl−). Cytokine levels of supernatants of ertumaxomab or trastuzumab cytotoxicity experiments were analyzed after 24 h. Supernatants containing ertumaxomab- or trastuzumab-induced cytokines were transferred to freshly plated HCT-8 cells (1 × 104). HCT-8 growth (○) was measured after a 3-d incubation period with the XTT proliferation assay. As a control was a sample of HCT-8 cells incubated with RPMI1640 medium, which was set as a standard of 100% HCT-8 growth; points, mean. Ab, antibody. D, HER2/neu specificity of ertumaxomab-induced cytotoxicity against HCT-8 cells: Cytotoxicity experiments were done in duplicates with 2 × 105 PBMC of 3 different healthy donors and an E/T ratio of 6:1. PBMC and target cells were preincubated with 200 ng/mL (▴), 500 ng/mL (○), or 2,000 ng/mL (X) of the anti-HER2/neu antibody 2502A followed by addition of ertumaxomab (100–0.001 ng/mL). Ertumaxomab-induced cytotoxicity was compared with samples without preincubation of 2502A (▪). Points, mean; bars, SD.

Jäger, M., Schoberth, A., Ruf, P., Hess, J., & Lindhofer, H. (2009). The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2. Cancer research, 69(10), 4270-4276.

Figure 5 Inhibition of ertumaxomab binding to HER2/neu by antibody 520C9.

A, FACS competition binding: inhibition of ertumaxomab (ertu; 1 μg/mL) binding to SK-BR-3 cells in the presence of varying ratios of 520C9 or trastuzumab (trastu) compared with ertumaxomab (10:1, 1:1, 1:10); all experiments were performed thrice; as control, the binding capacity of ertumaxomab, 520C9, and trastuzumab to SK-BR-3 cells was analyzed; points, mean; bars, SD. B, cytotoxicity of ertumaxomab (50 ng/mL) induced against HCT-8 cells (HER2 low, HER2 ampl−) after preincubation with a 100-fold higher concentration of trastuzumab (5,000 ng/mL). As controls were samples with trastuzumab (5,000 ng/mL) and ertumaxomab (50 ng/mL) alone. Cytotoxicity experiments were done in duplicate with 2 × 105 PBMC of 3 different healthy donors and a HCT-8 cell concentration of 1 × 104 (E/T, 20:1); sample: PBMC + HCT-8 + antibody; allogeneic reaction: PBMC + HCT-8 cells. Points, mean; bars, SD.

Jäger, M., Schoberth, A., Ruf, P., Hess, J., & Lindhofer, H. (2009). The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2. Cancer research, 69(10), 4270-4276.