NeuroMab™ Anti-AQP4 BBB Shuttle Antibody, Clone rAb-53

Figure 1 depicts differential binding of purified monoclonal NMO-IgG to M1 and M23 AQP4.

Representative fluorescence micrographs of rAb-53 and rAb-58 (green) binding as a function of concentration, and a reference AQP4 antibody (red).

Figure 2 depicts differential binding of purified monoclonal NMO-IgG to M1 versus M23 AQP4.

Binding curves of rAb-53 (left), rAb-58 (middle) and rAb-186 (right) to M1 vs. M23 AQP4 (mean ± SE, n=5). The curve representation fits the single-point binding model.

Figure 3 depicts the mechanism for increasing the binding affinity of NMO-IgG to array-assembled AQP4.

Prediction of bivalent versus monovalent binding mechanisms. AQP4 monomers (cylinders) are shown to assemble into tetramers (M1) or OAPs (M23). NMO-IgG (green) binds monovalently or bivalently to the (unknown) extracellular domain on AQP4.

Figure 4 depicts the mechanism for increasing the binding affinity of NMO-IgG to array-assembled AQP4.

Relative M1-M23 binding of whole IgG or purified Fab fragments of mouse anti-Myc (left), rAb-53 (middle) and rAb-58 (right) at fixed concentrations (mean ± SE, n=5).

Figure 5 depicts high affinity monoclonal, recombinant anti-AQP4 antibodies used in aquaporumab therapy.

Surface plasmon resonance measurements of recombinant antibody binding to AQP4 recombinant proteoliposomes showing the association/unbinding kinetics of different concentrations of rAb-53 (left) and fixed concentrations of different NMO rAbs (right).

Figure 6 depicts high affinity monoclonal, recombinant anti-AQP4 antibodies used in aquaporumab therapy.

Binding and unbinding kinetics of rAb-53 (25 μg/ml) to AQP4-expressing U87MG cells.

Figure 7 depicts that mutated, non-pathogenic rAb-53 (aquaporumab) blocks binding of pathogenic NMO-IgG to AQP4.

An irrelevant monoclonal NMO antibody and human NMO serum blocked the binding of Cy3-labeled rAb-53 to AQP4.

Figure 8 depicts that mutated non-pathogenic rAb-53 aquaporumab prevents CDC and ADCC in NMO-IgG exposed AQP4 expressing cells.

Live/dead assay of AQP4-expressing CHO cells exposed to human complement for 90 minutes along with control (non-NMO) mAb or rAb-53

Figure 9 depicts that mutant non-pathogenic rAb-53 aquaporumabs prevent CDC and ADCC in AQP4 expressing cells exposed to NMO-IgG.

Assays were performed as in A using complement + rAb-53 in the presence of 12.5 μg/ml of the indicated aquaporumab.

Figure 10 depicts that mutant non-pathogenic rAb-53 aquaporumabs prevent CDC and ADCC in AQP4 expressing cells exposed to NMO-IgG.

Live/dead cell assay after 60 min exposure to control (non-NMO) sera or NMO patient sera in the presence of complement and in the absence or presence of indicated aquaporumab.

Figure 11 depicts that mutant non-pathogenic rAb-53 aquaporumabs prevent CDC and ADCC in AQP4 expressing cells exposed to NMO-IgG.

ADCC assays use AQP4-expressing CHO cells incubated with NK cells along with control (non-NMO) mAb or rAb-53 or aquaporumab (alone) or rAb-53 and aquaporumab.