NeuroMab™ Anti-TLR4 BBB Shuttle Antibody(NRZP-1022-ZP2882)

Figure 1 is a graph depicting the binding of a murine monoclonal antibody (referred to herein as "18H10") to the TLR4/MD-2 complex.

Specificity of binding is shown by flow cytometry using mock transfected or TLR4/MD-2 transfected cells. The results using mock-transfected cells are shown in the filled graph (left), while the results using TLR4/MD-2 transfected cells are shown as in the outline graph (right).

Figure 2 is a graph depicting inhibition of lipopolysaccharide (LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells by monoclonal antibody mu18H10.

The cells were incubated with either mu18H10, HTA 125 (a commercially available anti-human TLR4 non-blocking MAb) or an antibody control at the indicated concentrations and subsequently incubated with LPS (15 ng/ml). IL-8 levels were assessed 16 hours post LPS treatment.

Figure 3 is a graph depicting inhibition of lipopolysaccharide (LPS)-induced IL-8 production by chimeric 18H10 MAb in TLR4/MD-2 transfected HEK 293 cells.

Cells were incubated with mu18H10, or chimeric 18H10 at the indicated concentrations and subsequently incubated with LPS (15 ng/ml). IL-8 levels were assessed 16 hours post LPS-treatment Inhibition of LPS-induced IL-8 production by the chimeric 18H10 was similar to the inhibition by the 18H10 mouse MAb of the present disclosure.

Figure 4 depicts inhibition of LPS-induced IL-8 production in human whole blood by monoclonal antibody mu18H10.

Whole blood was drawn from 3 healthy volunteers, treated with heparin and diluted 1:4 in RPMI medium. The following antibodies were added at the concentrations indicated: control monoclonal antibody; HTA125 and mu18H10. LPS was subsequently added for a final concentration of 10 ng/ml, and IL-8 levels were measured 6 hours post LPS treatment.

Figure 5 depicts the VH and VL nucleotide sequences of mu18H10 expressed as a chimeric MAb ("chimeric 18H10") is capable of specifically binding human TLR4/MD-2 complexes on the surface of transfected CHO cells.

MAb binding to the TLR4/MD-2 transfected CHO cells is shown by flow cytometry using chimeric 18H10 or an isotype matched control MAb at the concentrations indicated.

Figure 6 is a graph depicting the ability of hu18H10 humanized monoclonal antibody ("18H10 hum") to specifically bind human TLR4/MD-2 complexes on the surface of transfected CHO cells.

MAb binding to the TLR4/MD-2 transfected CHO cells is shown by flow cytometry using the hu18H10 antibody or the chimeric 18H10 ("18H10chim") (described above) at the concentrations indicated. Binding is measured as a cellular Mean Fluorescence Intensity (MFI) value.