Figure 1. IdeZ cleaves serum antibodies from multiple species.

(A) Schematic outlining IdeZ cleavage of IgG below the hinge region yielding multiple F(ab′)₂ and Fc fragments after reduction. (B) Serum samples from mouse, dog, primate, and human untreated (-) or treated (+) with recombinant IdeZ and analyzed by SDS-PAGE under reducing conditions. Gels were then stained with Coomassie blue. * indicates digested Fc/2 and Fd′ fragments. (C) Pooled human IgG untreated (-) or treated (+) with recombinant glutathione-S-transferase–IdeZ (GST-IdeZ) or commercial standard IdeZ and analyzed by SDS-PAGE under reducing conditions. Gels were then stained with Coomassie blue. IgG was cleaved by GST-IdeZ and IdeZ into multiple fragments as indicated. (D) Experimental timeline of in vivo GST-IdeZ dose optimization experiment. Mice were injected with pooled human IgG followed 24 hours later with no injection or injection with 3 different doses of GST-IdeZ. Blood serum samples were collected 72 hours post GST-IdeZ. Sac., sacrifice followed by tissue harvest. (E) Mice were injected IP first with pooled human IgG, followed by IV injection with PBS 24 hours later (-) or recombinant GST-IdeZ (1 mg/kg) (+). Blood samples were taken 72 hours after injection and analyzed by SDS-PAGE under reducing conditions with immunoblotting. IgG was probed with Fab- and Fc-specific antibodies. (F) Mice were injected IP first with pooled human IgG, followed by IV injection with PBS 24 hours later (-) or recombinant GST-IdeZ at 3 doses (0.25 mg/kg, 1 mg/kg, and 2.5 mg/kg) (+). Blood samples were taken 72 hours after injection and analyzed by SDS-PAGE under reducing conditions with immunoblotting. Human IgG was probed with an Fc-specific antibody. See complete unedited blots in the supplemental material.