Discussion
This finding underscores the necessity for careful evaluation of mutations during the engineering of protein therapeutics. The integration of multiple immunogenicity risk assessment tools offers critical insights for informed decision-making in candidate sequence design and therapeutic lead selection.
Methods
In this study, we utilized various immunogenicity risk assessment methods, including in silico prediction, dendritic cell internalization, MHC-associated peptide proteomics, in vitro HLA peptide binding, and in vitro T cell proliferation, to assess the immunogenicity risk of FLT3L-Fc variants.
Results
We identified a single point mutation in the human FLT3L-Fc protein that introduced highly immunogenic T cell epitopes, leading to the induction of T cell responses and thereby increasing the immunogenicity risk in clinical settings. Consequently, the variant with this point mutation was removed from further consideration as a clinical candidate.