Apley Kyle D, Bass Lindsay E, King Jaylyn, Downes Grant, Wang Kristen, Forchetti Mason V, Moore Daniel J, Kendall Peggy, Bonami Rachel H, Berkland Cory J
Department of Biomedical Engineering, Washington University, St. Louis, Missouri, 63130, USA.
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, 37232, USA.
Drug Deliv Transl Res. 2025 May 22. doi: 10.1007/s13346-025-01869-x.
Insulin-binding B cells are implicated in Type 1 Diabetes (T1D) pathology. Antigen-specific immunotherapy (ASIT) holds promise in T1D. However, ASIT-targeted suppression of insulin-binding B cells is hampered by insulin's hormonal activity and the resulting binding and endocytosis of insulin by insulin receptors (INSR). To evaluate ASIT strategies that target insulin-binding B cells in vivo, non-hormonally active insulin variants are needed. In this work, we aimed to improve upon prior non-hormonal insulin variants by making mutations to the insulin precursor, proinsulin, and including a c-terminal sortase (SrtA) tag (LPETGGHG) to enable facile site-selective bioconjugation to scaffolds or payloads. Of the insulin variants investigated that retained low-nM binding to the murine-derived insulin autoantibody mAb 125, proinsulin(F25D)-SrtA had the lowest INSR binding and activity and the greatest fibrillation resistance. Compared to desoctapeptide insulin, a previously proposed non-hormonal insulin variant, proinsulin(F25D)-SrtA demonstrated 50-fold lower INSR binding and 100-fold greater fibrillation lag time. However, insulin(F25D)-SrtA bound to the anti-insulin antibody 12M4 isolated from a presymptomatic T1D individual, whereas proinsulin(F25D)-SrtA and desoctapeptide insulin did not, highlighting the potential for anti-insulin B cells to develop in human T1D that would escape this ASIT moiety. The characteristics of proinsulin(F25D)-SrtA make it a well-suited non-hormonal insulin variant for insulin-binding B cell targeting and warrants additional study with other anti-insulin B cell specificities derived from T1D individuals.
胰岛素结合性B细胞与1型糖尿病（T1D）的病理过程有关。抗原特异性免疫疗法（ASIT）在T1D治疗中具有前景。然而，胰岛素的激素活性以及胰岛素受体（INSR）对胰岛素的结合和内吞作用阻碍了针对胰岛素结合性B细胞的ASIT靶向抑制。为了评估体内靶向胰岛素结合性B细胞的ASIT策略，需要非激素活性的胰岛素变体。在这项工作中，我们旨在通过对胰岛素前体胰岛素原进行突变，并添加一个c末端分选酶（SrtA）标签（LPETGGHG），以实现与支架或负载的简便位点选择性生物共轭，从而改进先前的非激素胰岛素变体。在所研究的与小鼠来源的胰岛素自身抗体mAb 125保持低纳摩尔结合的胰岛素变体中，胰岛素原（F25D）-SrtA具有最低的INSR结合和活性以及最大的抗纤维化能力。与先前提出的非激素胰岛素变体去八肽胰岛素相比，胰岛素原（F25D）-SrtA的INSR结合降低了50倍，纤维化延迟时间延长了100倍。然而，胰岛素（F25D）-SrtA与从症状前T1D个体分离出的抗胰岛素抗体12M4结合，而胰岛素原（F25D）-SrtA和去八肽胰岛素则不结合，这突出了在人类T1D中可能产生逃避这种ASIT部分的抗胰岛素B细胞。胰岛素原（F25D）-SrtA的特性使其成为一种非常适合靶向胰岛素结合性B细胞的非激素胰岛素变体，值得对来自T1D个体的其他抗胰岛素B细胞特异性进行进一步研究。
