Anti-Lewis Y Recombinant Antibody (MB311) (CAT#: TAB-0015CL)

Figure 1 Binding activity MB314 and MB311.

(A) Binding of MB311 and MB314 to the Lewis-Y positive tumor cell line SKBR-3 was determined by flow cytometry. Geometric Mean fluorescence intensity (MFI) was plotted vs. the logarithm of the antibody concentration and fitted using a sigmoidal four parameter fit using GraphPad Prism 4 software for calculation of EC50 values. Mean and SD of triplicates are shown. Binding of 100 μg/ml MB311 (dark gray line) or MB314 (black line) to purified human NK-cells expressing the FcγRIII variant V/F (B), to the FcγRIII variant F/F (C) and the FcγRIII variant V/V (D). Results obtained with medium only are shown as light gray lines.

Kircheis, R., Halanek, N., Koller, I., Jost, W., Schuster, M., Gorr, G., ... & Nechansky, A. (2012, July). Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314. In MAbs (Vol. 4, No. 4, pp. 532-541). Taylor & Francis.

Figure 2 MB314 and MB311 mediated effector functions.

CDC reactivity against two Lewis Y positive tumor cell lines was tested using a FACS based approach. Tumor cells were incubated with different concentrations of antibodies in the presence of human complement for 1 h. As comple-ment source frozen human serum was used. Cytotoxicity against SKBR-3 cells (A) or OVCAR-3 cells (B) was quantified by FACSTM analysis using a live/dead cell stain. Gated 7-AAD+ population (dead cells) as percentage of the whole population are plotted vs. the logarithm of the antibody concentra-tion and fitted using a sigmoidal four parameter fit using GraphPad Prism 4 software. Mean and SD of triplicates are shown.

Kircheis, R., Halanek, N., Koller, I., Jost, W., Schuster, M., Gorr, G., ... & Nechansky, A. (2012, July). Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314. In MAbs (Vol. 4, No. 4, pp. 532-541). Taylor & Francis.

Figure 3 Intracellular Expression of tumor necrosis factor (TNF) by CD14+ cells, but not by CD3+ or CD56+ cells.

SKBR-3 target cells and effector cells (PBMCs from a healthy volunteer donor) at an E:T ratio of 25:1 were incubated with 74 ng/ml of MB311 for 5 h. Cells were harvested, stained with FITC-mouse anti-CD14 antibody or mouse anti- CD56 antibody and anti-mIgG Dylight 488, or Cy5-mouse anti-CD3 antibody, fixed, permeabilized and subsequently stained with PE-mouse anti human TNFα antibody. Staining was measured by FACS analysis. The data reflect gating on PBMC effector cells (red) (and excluding target cells, green) based on forward and side scattered light signals (left part). Intracellular staining of TNFα gated on CD14+ or CD56+ (middle part) and CD3+ cells are shown (right part).

Kircheis, R., Halanek, N., Koller, I., Jost, W., Schuster, M., Gorr, G., ... & Nechansky, A. (2012, July). Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody