734-P: Cellular Internalization and Localization of Once-Weekly Basal Insulin Fc (BIF)

Abstract

Basal Insulin Fc (BIF, LY3209590) is an insulin Fc-fusion protein in clinical testing as a once weekly treatment for diabetes mellitus. BIF is comprised of a human single-chain insulin fused to a human IgG2 Fc domain through a peptide linker. The cellular internalization characteristics of BIF compared to native insulin using U2OS cells expressing human insulin receptor (hIR) are reported in this work. Internalization and sub-cellular localization were visualized by immuno-fluorescent confocal microscopy in U2OS cells expressing human insulin receptor. Internalization of BIF was observed, and the insulin and Fc moieties remained co-localized. Co-localization studies using an antibody to the early endosomal marker Early Endosome Antigen 1 (EEA1) showed that human insulin and BIF (insulin and Fc moieties) were rapidly internalized and co-localized with EEA1. During ligand washout, the remaining insulin and Fc moieties of BIF remained largely co-localized with EEA1 and a concomitant loss of immunostaining was observed, similar to human insulin. Studies using an antibody to the lysosomal marker Lysosomal Associated Membrane Protein-1 (LAMP-1) revealed a low level of lysosomal localization for human insulin and BIF (insulin and Fc moieties), indicating a similar intracellular trafficking pattern of human insulin and BIF to the lysosomal degradative pathway. The co-localization of remaining ligand decreased over time. IR internalization and transport to early endosomes was measured using an enzyme complementation assay. The EC50 for BIF was 4.7 ± 2.1 nM (n = 3), and the EC50 for human insulin was 0.06 ± 0.01 nM (n = 3). In summary, BIF stimulated IR internalization to a similar maximum level; however, with decreased potency versus human insulin. The subcellular trafficking pattern of BIF is similar to human insulin. BIF undergoes rapid internalization and transport to early endosomes with limited transport to the lysosomes and undergoes loss of cellular immunostaining during ligand washout. Disclosure C. Volk: None. C. Zhang: Employee; Self; Eli Lilly and Company. J. S. Moyers: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. Funding Eli Lilly and Company