Anti-human Integrin α5β1 Recombinant Antibody (MOR04624) (TAB-723LC) (CAT#: TAB-723LC)

Figure 1 Fab-mediated dose-dependent inhibition of K562 cells to fibronectin Anti human α5β1-specific Fab were tested for their ability to inhibit the binding of fluorescent dye-loaded K562-cells to immobilized fibronectin.

After adhesion, cells were lysed and fluorescence was determined as a measure for adherent cells. Fibronectin alone indicates the maximum adhesion whereas the overall background of the assay was determined on BSA-coated cells.

Figure 2 Anti α5β1-function blocking antibodies induce apoptosis in endothelial cells.

The induction of caspase 3/7 activation of the purified Fab in the monovalent format was determined using HUVEC cells in serum free endothelial cell medium. Caspase activity was determined using a commercially available chemoluminescent assay system (Caspase GIo, PROMEGA) according to the manufacturers' instructions.

Figure 3 Competition FACS of Fab and IIA1.

The FACS-competition indicates that MOR04624 competes with the epitope of the reference antibody IIA1 on HT29α5 cells. It can be concluded that both antibodies share the similar epitope, whereas all other Fab react with unrelated binding sites on the α5β1 integrin. (black line-Fab binding, green line-Fab binding when competed with reference antibody IIA1).

Figure 4 Affinity-matured anti α5β1-function blocking antibodies potently induce apoptosis in endothelial cells.

The induction of caspase 3/7 activation of the purified Fab in the monovalent format was determined using HUVEC cells in serum free endothelial cell medium. Caspase activity was determined using a commercially available chemoluminescent assay system (Caspase GIo1 PROMEGA) according to the manufacturers' instructions.

Figure 5 Optimized IgGs in HUVEC adhesion assay.

Inhibition of adhesion of HUVEC cells to fibronectin by α5β1 function blocking IgG antibodies. IgG MOR04974, MOR04975, MOR04977, MOR04985 block adhesion with a similar IC50 than IIA1. Conversion from Fab to IgG resulted in an approximately 2-fold improvement.

Figure 6 HUVEC viability assay-analysis of anti-α5β1 integrin IgGs.

Inhibition of viability of HUVEC cells by α5β1 function blocking IgG antibodies. HUVEC cells were plated on fibronectin coated plates, incubated with increasing concentration of IgG antibodies and survival measured after 48 h. IgG MOR04974, MOR04975, MOR04977, MOR04985 block adhesion with a similar IC50 than IIA1. Conversion from Fab to IgG resulted in an approximately 2-fold improvement.

Figure 7 HUVEC Caspase assay of anti-α5β1 integrin IgGs.

The induction of Caspase 3/7 activation by the α5β1 function blocking IgG antibodies was determined using HUVEC cells in serum free endothelial cell medium. Caspase activity was determined using a commercially available chemoluminescent assay system (Caspase glo, PROMEGA) according to the manufacturers' instructions. MOR04974, MOR04975, MOR04977 and MOR04985 are similar active as the reference antibody IIA1.