High-throughput Western Blot for in vitro and in vivo assessment of human Ig specific cleaving enzymes

Abstract

Abstract Antibodies play a vital role in the body’s immune defense mechanism. However, pathogenic Ig antibodies are thought to contribute to several autoimmune conditions and transplant rejection. Proteases derived from human pathogens can specifically cleave IgG into F(ab’)2 and Fc fragments, thus allowing for rapid clearance of IgG. This unique trait suggests a novel opportunity to use these molecules to treat autoantibody mediated diseases. Using our IMPACT platform leveraging machine learning, we have engineered Ig proteases that specifically cleave IgG, and treatment with these proteases causes cleavage below the hinge region, which can result in the generation of the following degradation products: 1) Fab’2 and Fc fragments lacking effector function; or 2) single chain cleaved fragments with reduced effector functions. To detect these species, we developed a method using JESS, an automated high throughput western blot instrument. Using this system, we can detect intact, single cleaved and fully cleaved IgG in a time and dose dependent manner in both in vitro and in vivo assays. In conclusion, we successfully optimized a high throughput assay format that is highly specific and sensitive in evaluating the cleavage activity of different Ig proteases and could be a powerful tool to assess efficacy of IgG cleaving enzymes in the clinic.