D001 Development of an HTT exon1-selective MSD immunoassay with novel HTT P90 neo-epitope-specific antibodies

<h3></h3> Recent evidence suggests that an exon 1 protein fragment of mutant huntingtin (HTT) is produced by the translation of a misspliced form of HTT RNA. Immunoassays currently available to support HTT-lowering therapeutic approaches are not able to effectively measure HTT exon1 (1-90) due to the lack of neo-epitope P90 selective antibodies. One of the published assays for HTT exon1 employs 2B7 (HTT N-terminal antibody) and MW8 (antibody raised against an aggregate of HTT exon1 with 67Q). To improve the selectivity and sensitivity of the assay for HTT Exon1, we generated two recombinant antibodies that recognize the C-terminal proline at position 90 (P90) of HTT exon1 protein and paired them with 2B7 for the MSD immunoassay development. The performance of these MSD immunoassays was assessed using recombinant proteins, including HTT full-length protein and HTT exon1 fragments. We observed a robust increase in MSD assay signal response with increasing concentrations of recombinant HTT exon1 protein and an absence of signal for the negative controls, demonstrating the selectivity of this novel P90 MSD immunoassay for HTT exon1. A comparison with the 2B7/MW8-based MSD assay showed that the new P90 antibody-based MSD immunoassay outperformed in terms of sensitivity, selectivity, and dynamic range. Additionally, age-dependent decrease in HTT exon1 levels in the cortex and striatum of the HD mouse model Q175 were confirmed using the novel P90 MSD immunoassay. Currently, the qualification of the MSD assay shows good promise and efforts are underway to port it to an ultra-sensitive platform for use in human biosamples.