Dan Qin ¹ Qui Phung ¹ Patrick Wu ¹ Zhaojun Yin ¹ Sien Tam ¹ Peter Tran ¹ Adel M Elsohly ¹ Joshua Gober ¹ Zicheng Hu ¹ Zhenru Zhou ¹ Sivan Cohen ¹ Dongping He ¹ Travis W Bainbridge ¹ Christopher C Kemball ¹ Jonathan Zarzar ¹ Alavattam Sreedhara ¹ Nicole Stephens ² Jérémie Decalf ¹ Christine Moussion ¹ Zhengmao Ye ² Mercedesz Balazs ¹ YINYIN LI ^1*

• ¹ Genentech Inc., San Francisco, United States
• ² Gilead (United States), Foster City, California, United States

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 immunogenicity risk of FLT3L-Fc variants.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 decisionmaking in candidate sequence design and therapeutic lead selection.