Abstract
Major histocompatibility complex class II (MHCII)-restricted antigen priming of CD4+ T cells is both involved in adaptive immune responses and the pathogenesis of autoimmune diseases. Degradation of invariant chain Ii, a protein that prevents premature peptide loading, is a prerequisite for nascent MHCII–peptide complex formation. A key proteolytic step in this process is mediated by cathepsin S. Inhibition of this cysteine protease is known to result in the intracellular accumulation of Lip10 in B cells. Here, we describe the development and application of a neoepitope-based flow cytometry assay measuring accumulation of Lip10. This novel method enabled the investigation of cathepsin S-dependent MHCII maturation in professional antigen-presenting cell (APC) subsets. Inhibition of cathepsin S by a specific inhibitor, RO5459072, in human PBMC ex vivo resulted in accumulation of Lip10 in B cells and myeloid dendritic cells, but not in plasmacytoid dendritic cells and only to a minor degree in monocytes. We qualified Lip10 as a pharmacodynamic biomarker by showing the cathepsin S inhibitor-dependent accumulation of Lip10 in vivo in cynomolgus monkeys treated with RO5459072. Finally, dosing of RO5459072 in a first-in-human clinical study (www.ClinicalTrials.gov, identifier NCT02295332) exhibited a dose-dependent increase in Lip10, confirming target engagement and demonstrating desired pharmacologic inhibition in vivo. The degree of cathepsin S antagonist-induced maximum Lip10 accumulation in APCs varied significantly between individuals both in vitro and in vivo. This finding has not been reported previously using alternative, less sensitive methods and demands further investigation as to the potential of this biomarker to predict response to treatment. These results will help guide subsequent clinical studies investigating the pharmacokinetic and pharmacodynamic relationship of cathepsin S inhibitor RO5459072 after multiple dosing.
Highlights
Major histocompatibility complex (MHC) class II (MHCII) molecules are central to adaptive immune responses
Synthesized Major histocompatibility complex class II (MHCII) molecules are initially associated with the CD74 invariant chain (Ii), a chaperone molecule required for the maturation of the MHCII complex by facilitating appropriate folding, preventing premature peptide loading and directing nascent MHCII molecules to the late endosomal compartment for peptide loading [7, 8]
We show the application of this assay as a tool to investigate the requirement of cathepsin S for MHCII maturation in antigen-presenting cell (APC) subpopulations
Summary
Major histocompatibility complex (MHC) class II (MHCII) molecules are central to adaptive immune responses. Synthesized MHCII molecules are initially associated with the CD74 invariant chain (Ii), a chaperone molecule required for the maturation of the MHCII complex by facilitating appropriate folding, preventing premature peptide loading and directing nascent MHCII molecules to the late endosomal compartment for peptide loading [7, 8]. The proteolysis of Ii occurs as a series of sequential steps, leaving the class II-associated invariant chain peptide (CLIP), a 24-amino-acid fragment, in the peptide-binding cleft of MHCII molecules (Figure 1A) [9, 10]. The MHC-like molecule HLA-DM facilitates the exchange of CLIP with 10–14 amino acid long peptides within the endolysosomal compartment, after which the MHCII–peptide complex is transported to the cell surface for presentation of the peptide antigen to CD4+ T cells
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