Human Anti-AXL Recombinant Antibody (clone h#11B7-T11) (CAT#: HPAB-0116-LS)

Figure 1 ELISA experiments to investigate the effects of humanized 11B7 anti-Axl antibodies on AxI receptor phosphorylation.

Hs578T breast cancer cells (top) and NCI-H292 lung cancer cells (bottom) were starved, pre-incubated with no antibody or 10 mg/ml of Gammagard control antibody as well as the humanized anti-Axl antibodies 11 B7-T5, 11B7-T6, 11 B7-T11, 11B7-T23, and 11 B7-T25, treated with or without 400 ng/ml mGasθ, and lysed. Lysates were transferred to anti-phospho-tyrosine antibody 4G10-coated Maxi-Sorp 96 well plates, which then were washed and incubated with 0.125 mg/ml biotinylated rat anti-Axl antibody 12B7, AP-conjugated streptavidin and AttoPhos substrate solution in order to collect fluorescence intensities. See text for details. Compared with Gammagard control antibody, the humanized anti-Axl antibodies 11 B7-T5, 11 B7-T6, 11 B7-T11, 11 B7-T23, and 11B7-T25 significantly reduced Gas6-mediated AxI activation in both cell lines as indicated by decreased AxI tyrosine phosphorylation levels in Gas6-stimulated versus corresponding non-stimulated cells.

Figure 2 FACS analysis to investigate the effects of humanized 11B7 anti-Axl antibodies on AxI receptor internalization.

Hs578T breast cancer cells were starved, incubated with no antibody or 10 mg/ml of Gammagard control antibody as well as the humanized anti-Axl antibodies 11B7-T5, 11 B7-T6, 11B7-T11, 11 B7-T23, and 11 B7-T25 for the indicated periods of time, and fixed with formaldehyde. Cells were stained with rat anti-Axl mAb 2A1 primary antibody and PE-conjugated donkey-anti-rat IgG secondary antibody, and subjected to FACS analysis. See text for details. In contrast to Gammagard control antibody, treatment of Hs578 breast cancer cells with the humanized anti-Axl antibodies 11 B7-T5, 11B7-T6, 11B7-T11, 11B7-T23, and 11B7-T25 leads to internalization of the AxI receptor. Relative AxI expression levels in %, defined as mean fluorescence intensity of anti-Axl mAb treated samples relative to untreated samples of the respective treatment period is shown.

Figure 3 Spheroid-based cellular angiogenesis assay to investigate the effects of humanized 11B7 anti-Axl antibodies on Gas6-induced endothelial cell sprouting.

VEGF-A pre-treated HUVEC spheroids were embedded in a 3D collagen gel, stimulated with 1 mg/ml human Gas6 and treated with indicated concentrations of Gammagard control antibody as well as the humanized anti-Axl antibodies 11B7-T11 for 24 h. The mean+SEM of the cumulative sprout length of 10 randomly selected spheroids per data point was analyzed (left panel) and the relative inhibition by the antibody was determined (right panel). Fitting of IC50 curves and calculation of IC50 values was performed with GraphPad Prism 4.03.

Figure 4 Binding activity of the rat anti-human AXL humanized antibodies h#11B7-T7 to h#11B7-T13 determined by ELISA using human AXL-Fc coated plate.