Background: Therapeutic de novo dystrophin has the potential to elicit a humoral immune response (anti-dystrophin antibodies) as a neo-antigen, although any treatment-emergent anti-dystrophin antibodies may or may not be pathogenic. As a potential safety signal, all approved dystrophin replacement therapies show post-marketing commitments to screen treated patients for treatment-emergent anti-dystrophin antibodies. The method published by FDA, and typically preferred by regulators, is called Luciferase immunoprecipitation system (LIPS).1 This method uses fusion proteins of the protein target to luciferase. When the luciferase-target fusion protein is mixed with patient sera, potential anti-target human antibodies bind, the antibody-fusion complexes are immunoprecipitated with Protein A/G resin, and luciferase activity assayed.
Objective: To develop a validated, regulatory-accepted LIPS assay for the viltolarsen/Viltepso program that is supportive of the post-marketing program.
Methods: Eight luciferase-dystrophin fusion protein expression vectors were constructed and tested for suitability for expression in COS1 cells, and performance in the LIPS assay. We tested 4 dystrophin variants (full length dystrophin [427kDa] and 3 overlapping subfragments [exons 1-30, exons 30-52, exons 52-79]), each with nanoluciferase (Nluc) fused to both the amino- and carboxyl-termini. While all 8 dystrophin fusion constructs were shown to successfully express the expected fusion proteins after transfection in COS1 cells, the 4 carboxyl-terminal Nluc fusions expressed at higher levels and were carried forward. Three region-specific positive control anti-dystrophin antibodies were tested with each of the fusion constructs for performance in the LIPS assay.
Results: Full-length dystrophin was not efficiently pulled down by any antibody, whereas the 3 overlapping dystrophin subfragments were efficiently pulled down (immunoprecipitations). Each of the 3 overlapping subfragments and paired antibodies were validated in regulatory-accepted LIPS assays. A population-based cut-point was also established for each assay.
Conclusion: A regulatory-accepted LIPS assay was established for three overlapping regions of the complete dystrophin protein.