INTRODUCTION: As a crucial asset for human health and modern medicine, an increasing number of biotherapeutics are entering the clinic. However, due to their complexity, these drugs have a higher potential to be immunogenic, leading to the generation of anti-drug antibodies (ADAs). Clinically significant ADAs have an impact on pharmacokinetics (PK), pharmacodynamics (PD), effectiveness, and/or safety. Thus, it is crucial to understand, manage and minimize the immunogenicity potential during drug development, ideally starting from the molecule design stage. 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. 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.
Qin D, Phung Q, Wu P, Yin Z, Tam S, Tran P, ElSohly A M, Gober J, Hu Z, Zhou Z, Cohen S, He D, Bainbridge T W, Kemball C C, Zarzar J, Sreedhara A, Stephens N, Decalf J, Moussion C, Ye Z, Balazs M, and Li Y. (2025) A single point mutation on FLT3L-Fc protein increases the risk of immunogenicity. Front Immunol 16(1):1519452 . [article]