Design and characterization of a novel human Granzyme B inhibitor

The intracellular roles of Granzyme B (GrB) in immune-mediated cell killing have been extensively studied. Recent data also implicate GrB in extracellular pathways of inflammation, cytokine activation and autoimmunity. Targeting (GrB) provides a new pharmaceutical agent for various inflammatory disorders. Serpina3n is a mouse extracellular inhibitor of GrB. There is no apparent equivalent in humans. In this study, we used a novel applied genetics approach to engineer a new extracellular GrB serpin. A chimeric protein was generated in which the reactive center loop (RCL) of human extracellular antichymotrypsin (ACT) was replaced with that of serpina3n. This serpin contained 27 amino acid residues from the serpina3n RCL and the remaining 395 residues from human ACT. The insertion converted human ACT into a GrB-inhibitory serpin. Several critical residues were identified by scanning mutagenesis on the chimera and serpina3n. Targeted mutagenesis was conducted on wild-type human ACT by specifically substituting those critical residues, creating a novel inhibitor that contains 99.3% human ACT sequence with only three point mutations. Wild-type human ACT had a k(ass) for GrB of 2.26 x 10(4) M-1 s(-1), whereas the novel inhibitor binds GrB with a k(ass) of 7.65 x 10(5) M-1 s(-1). This new drug candidate can be developed in animal models and further tested in clinical trials to help us understand the role of GrB in numerous disorders.