NeuroMab™ Anti-HMGB1 BBB Shuttle Antibody, Clone NR304P

Figure 1 shows the binding ability of #10-22 chimeric antibody to HMGB1 evaluated after humanization.

This assessment was performed by ELISA assay using HMGB1 immobilized immunoplates. As a control, using the H chain of #10-22 chimeric antibody, assuming that the OD value of this control is 100%, the binding rate of #10-22 humanized antibody (EV007156) to HMGB1 was calculated.

Figure 2 shows the evaluated binding ability of anti-HMGB1 antibodies (#10-22 chimeric antibody, EV007156, S6 and G4) to recombinant HMGB1 (derived from Sf9 cells).

Figure 3 shows the inhibitory effect of anti-HMGB1 antibodies on RAGE binding.

After the amount of HMGB1 was quantified from the OD value obtained by ELISA, the binding rate of HMGB1 to RAGE was calculated assuming that the amount of HMGB1 (2 μg/ml) used for the binding inhibition assay was set as 100%. Open circle: #10-22 chimera, closed square: EV007156, open square: G4, closed circle: control Ig.

Figure 4 shows the inhibitory effect of anti-HMGB1 antibodies on HMGB1-induced TNF-α releasing activity in PBMCs.

PBMC obtained from peripheral blood were stimulated with HMGB1 and the amount of TNF-α contained in the culture supernatant obtained after 24 hours was quantified (eBioscience, Human TNF-alpha Ready-Set-Go!). Filled circle: negative control (EV2001), filled triangle: EV007156, asterisk: S6, open circle: G4.

Figure 5 shows the pharmacokinetic test results of EV007156 in mice.

EV007156 was intraperitoneally administered to C57BL/6N mice at a dose of 10 mg/kg, and blood samples collected at 0.25, 3, 7, 14 and 24 days after administration were quantified by ELISA for EV007156 contained therein.

Figure 6 shows the Western blotting epitope mapping of EV007156.

Full-length HMGB1 and single deletion constructs were electrophoresed on polyacrylamide gels and detected with EV007156.

Figure 7 shows the death protection effect of EV007156 administered to sepsis model mice.

EV007156 was administered to CLP-treated mice at a dose of 10 mg/kg, and their survival rate was calculated until 6 days after surgery. Solid circle: negative control group, solid triangle: EV007156 administration group.

Figure 8 shows the ability of the antibody to bind to HMGB1 evaluated after humanization of the L chain of the #10-22 chimeric antibody.

This assessment was performed by ELISA assay using HMGB1 immobilized immunoplates. Using #10-22 chimeric antibody as a control, calculate the binding rate (H chain) of the antibody obtained by co-expressing #10-22 (L chain) humanized antibody (EV007156L) and #10-22 chimeric antibody to HMGB1, Suppose the OD value of this control is set to 100%.