Anti-Human EGFR Recombinant Antibody (Zalutumumab)

Figure 1 Development of a panel of EGFR-specific mAbs with distinct functionalities.

A, Binding of zalutumumab, zalu-N297Q, mAb 018, and mAb 018-N297Q to cell-associated EGFR on A431 cells analyzed by flow cytometry; data were analyzed using a four-parameter logistic curve fit. Shown is the mean fluorescence intensity (MFI). B, The capacity of the different mAbs to induce ADCC analyzed in a 51Cr-release assay using human PBMCs as effector cells and A431 cells as target cells. Data shown are percentages specific lysis, calculated as described in Materials and Methods. C, mAb-induced inhibition of EGFR phosphorylation in A431 cells upon stimulation with EGF measured in an EGFR phosphorylation inhibition assay. Data shown are time-resolved fluorescences (TRF). D, Inhibition of A431 cell proliferation determined in a proliferation inhibition assay using Alamar Blue staining. Each data point represents mean ± SEM of triplicates, and experiments shown are representative of at least two independent experiments.

Overdijk, M. B., Verploegen, S., van den Brakel, J. H., van Bueren, J. J. L., Vink, T., van de Winkel, J. G., ... & Bleeker, W. K. (2011). Epidermal growth factor receptor (EGFR) antibody-induced antibody-dependent cellular cytotoxicity plays a prominent role in inhibiting tumorigenesis, even of tumor cells insensitive to EGFR signaling inhibition. The Journal of Immunology, 1003926.

Figure 2 Zalutumumab and mAb 018 are functional in an ADCC assay with murine effector cells.

The capacity of the different mAbs to induce ADCC was analyzed in a 51Cr-release assay using mouse bone marrow-derived macrophages as effector cells, and A431 cells as target cells. mAbs were tested at a concentration of 10 μg/ml with six replicates. Data represent mean ± SEM of three independent experiments.

Overdijk, M. B., Verploegen, S., van den Brakel, J. H., van Bueren, J. J. L., Vink, T., van de Winkel, J. G., ... & Bleeker, W. K. (2011). Epidermal growth factor receptor (EGFR) antibody-induced antibody-dependent cellular cytotoxicity plays a prominent role in inhibiting tumorigenesis, even of tumor cells insensitive to EGFR signaling inhibition. The Journal of Immunology, 1003926.

Figure 3 Both zalutumumab and mAb 018 are capable of inhibiting growth of tumor cells expressing mutant KRAS.

Eight mice per group were inoculated s.c. with 1 × 106 A431-KRAS4bG12V cells, which are insensitive for EGFR signaling inhibition. A, Single treatment at day 0 with 5 mg/kg mAb. B, Single treatment at day 0, at indicated dose levels. Data shown are mean tumor volumes ± SEM.

Overdijk, M. B., Verploegen, S., van den Brakel, J. H., van Bueren, J. J. L., Vink, T., van de Winkel, J. G., ... & Bleeker, W. K. (2011). Epidermal growth factor receptor (EGFR) antibody-induced antibody-dependent cellular cytotoxicity plays a prominent role in inhibiting tumorigenesis, even of tumor cells insensitive to EGFR signaling inhibition. The Journal of Immunology, 1003926.

Figure 4 Histology and immunohistochemistry of biopsies from zalutumumab-injected skin treated with HuMab-10F8 or untreated.

Data shown are representative sections of skin biopsies of one subject for part III of the study. Biopsies were taken at day 14 after two injections with 100 µg of zalutumumab with [A, C, E, G, I, K] or without HuMab-10F8 [B, D, F, H, J, L]. A, B: HE staining; C, D: staining for EGFR; E–H: staining for macrophages and I–L: staining for neutrophils. Extensive influx of inflammatory cells in the vicinity of the hair follicle with histological signs of folliculitis was observed in zalutumumab-injected skin (right panels), whereas in the zalutumumab-injected skin treated with HuMab-10F8 (left panels) inflammatory cells were virtually absent and sebaceous glands and hair follicles showed normal histological features. Inflammatory infiltrate in the zalutumumab-injected skin was mainly composed of neutrophils (neutrophil elastase positive cells, J, L) and to a lesser extent of macrophages; (CD68 positive cells F, H). Scale bars are 100 µm (A–F, I, J) and 20 µm (G, H, K, L). G, H, K and L are magnifications of the squared areas indicated in E, F, I and J.

Bangsgaard, N., Houtkamp, M., Schuurhuis, D. H., Parren, P. W., Baadsgaard, O., Niessen, H. W., & Skov, L. (2012). Neutralization of IL-8 prevents the induction of dermatologic adverse events associated with the inhibition of epidermal growth factor receptor. PLoS One, 7(6), e39706.

Figure 1 Anti-Human EGFR Antibody (TAB-040) in ELISA

ELISA analysis of TAB-040 was performed with EGFR Protein, Human, Recombinant (His Tag).
The secondary antibody: HRP-Anti-Human IgG (H+L)

Figure 2 Anti-Human EGFR Antibody (TAB-040) in WB

Western blot analysis of TAB-040 was performed with EGFR Protein, Human, Recombinant (His Tag).
TAB-040 incubation concentration: 2ng/μL.
The secondary antibody: HRP-Anti-Human IgG (H+L)
Lane 1: Reducing antigen (0.6μg)

Figure 3 Anti-Human EGFR Antibody (TAB-040) in DB

Dot blot analysis of TAB-040 was performed with EGFR Protein, Human, Recombinant (His Tag).
TAB-040 incubation concentration: 2ng/μL.
NC: Negative control PC: Positive control