NeuroMab™ Anti-TLR4 BBB Shuttle Antibody(NRZP-1022-ZP2883)

Figure 1 is a graph depicting the binding of a murine monoclonal antibody (referred to herein as "15C1") 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 mu15C1.

The cells were incubated with the mu15C1 monoclonal antibody, HTA 125 (anti-human TLR4 non-blocking monoclonal antibody) and an isotype-matched control (IgG1) at the indicated concentrations and subsequently incubated with LPS (15 ng/ml). IL-8 levels were assessed 16 hours post LPS treatment.

Figure 3 depicts the inhibition of LPS-induced IL-6 production in human whole blood by monoclonal antibody mu15C1.

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: Isotype matched control (IgG1); HTA125 (anti-human TLR4 non-blocking monoclonal antibody); mu15C1 and 28C5 (anti-human CD14 blocking monoclonal antibody). LPS was subsequently added for a final concentration of 10 ng/ml.

Figure 4 is a graph depicting the VH and VL nucleotide sequences of mu15C1 expressed as a chimeric MAb ("chimeric 15C1") is able to specifically bind human TLR4/MD-2 complexes on the surface of transfected CHO cells.

MAb binding to the TLR4/MD-2 complex is shown by flow cytometry using chimeric 15C1 or an isotype matched control monoclonal antibody at the indicated concentration.

Figure 5 is a graph depicting inhibition of lipopolysaccharide (LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells by chimeric 15C1 MAb.

Cells were incubated with mu15C1 or chimerical 15C1 at the concentrations indicated 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 15C1 was similar to the inhibition by the mu15C1 mouse MAb of the present disclosure.

Figure 6 is a graph showing that the hu15C1 humanized antibody can specifically bind to the human TLR4/MD-2 complex on the surface of transfected CHO cells.

MAb binding to the TLR4/MD-2 transfected CHO cells is shown by flow cytometry using the hu15C1 antibodies or the chimeric 15C1 ("15C1 CHIM") (described above) at the concentrations indicated. Binding is measured as a cellular Mean Fluorescence Intensity (MFI) value.

Figure 7 depicts inhibition of LPS-induced IL-6 production in human whole blood by monoclonal antibody hu15C1. hu15C1 4-28/A26 was compared to an isotype-matched control (IgG1) and the 15C1 chimeric antibody described here.