Abstract - Kyle Apley

Insulin variants to selectively target insulin-specific B cells: Enabling antigen-specific immunotherapy for type 1 diabetes

Kyle Apley1, Jaylyn King2, Grant Downes3, Danny Griffin1, Cory Berkland1, 3, Mark Farrell4

1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS;  2Department of Molecular Biosciences, University of Kansas, Lawrence, KS;  3Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS;  4Department of Medicinal Chemistry, University of Kansas, Lawrence, KS

B cells are a promising target for type 1 diabetes (T1D) immunotherapy as evidence points to their role in the loss of immune tolerance. However, clinical trials for B cell-depletion have relied on globally immunosuppressive therapies that increase the risk of infection. Therefore, our goal is to design and evaluate molecular antigen-specific immunotherapies to eliminate or restore tolerance in autoreactive B cells for T1D while avoiding global immunosuppression. Here, we share the development of insulin variants to selectively target insulin-specific B cells by incorporating mutations to reduce binding to hormonal insulin-receptors, a sortase tag to enable site-specific conjugation, and mutations to reduce self-association for the construction of multi-valent conjugates. Insulin-variants were expressed in E. coli, biophysically characterized, and conjugated with functionalized oligoglycine peptides using an optimized sortase-mediated ligation method. The binding selectivity of the insulin variants for anti-insulin B cells is evaluated by flow cytometry analysis of fluorescently-labeled insulin variants with splenocytes possessing an anti-insulin B cell receptor heavy chain transgene (VH125). The potential of the insulin variants for payload delivery is demonstrated by delivery of MMAF, a microtubule inhibitor, to anti-insulin NOD mouse B cells with an IC50 concentration 100-fold lower than for NOD mouse B cells. These data support the insulin variants as promising targeting ligands for anti-insulin B cells and warrant further characterization and biological experimentation.