D01 Two independent validations of a mutant huntingtin protein (MHTT) assay to support the clinical development of mHTT-targeting therapies in HD

Background Huntington’s disease (HD) is caused by a CAG repeat expansion in the huntingtin gene, resulting in the production of toxic mutant huntingtin protein (mHTT). Quantification of mHTT in the cerebrospinal fluid (CSF) of patients with HD using a method validated as per international guidelines is critical to support the clinical development of mHTT-targeting therapies. Aims To validate a bioanalytical method for quantifying relative mHTT levels in human CSF in two independent laboratories. Methods The assay was optimised in a single laboratory before its transfer to a second independent laboratory. All results were generated in regulated bioanalytical environments (i.e. by Good Clinical Practice-trained personnel in Good Laboratory Practice-certified laboratories) using a bead-based sandwich ligand-binding assay with Single Molecule Counting detection on the SMCxPROTM (Merck). The ultra-sensitive assay employs the antibody pair 2B7/MW1 for capture and detection and artificial CSF as a surrogate matrix. Assay validation followed international guidelines adapted to the context of use. Results Full assay validation performed in two independent laboratories (Roche, PRA Health Sciences) confirmed robust inter- and intra-assay accuracy and precision and a high sensitivity (lower limit of quantification =1.63 pg-eq/mL [Roche], 1.64 pg-eq/mL [PRA Health Sciences]). Parallelism and specificity experiments involving CSF from patients with HD as well as artificial CSF matrices confirmed the selectivity and specificity of the assay, and the absence of a matrix effect. Stability of mHTT in artificial CSF was found to be sufficient to accommodate bioanalysis. Conclusions The independent method validations demonstrate that this ultra-sensitive assay can be replicated and transferred, making it a reliable and broadly relevant tool for generating biomarker data in registrational clinical trials for mHTT-lowering therapies. Study sponsored by F. Hoffmann-La Roche Ltd.