Sung-Ting Chuang Oscar Alcazar Brandon Watts Midhat Abdulreda ^* Peter Buchwald ^*

• University of Miami, Coral Gables, Florida, United States

As part of our work to develop small-molecule inhibitors (SMIs) of the CD40-CD40L(CD154) costimulatory protein-protein interaction, here, we describe the ability of two of our most promising SMIs, DRI-C21041 and DRI-C21095, to prolong the survival and function of islet allografts in two murine models of islet transplantation (under the kidney capsule and in the anterior chamber of the eye) and to prevent autoimmune type 1 diabetes (T1D) onset in NOD mice. In both transplant models, a significant portion of islet allografts (50%-80%) remained intact and functional long after terminating treatment, suggesting the possibility of inducing operational immune tolerance via inhibition of the CD40-CD40L axis. SMI-treated mice maintained the structural integrity and function of their islet allografts with concomitant reduction in immune cell infiltration as evidenced by direct longitudinal imaging in situ. Furthermore, in female NODs, three-month SMI treatment reduced the incidence of diabetes from 80% to 60% (DRI-C21041) and 25% (DRI-C21095). These results (i) demonstrate the susceptibility of this TNF superfamily protein-protein interaction to smallmolecule inhibition, (ii) confirm the in vivo therapeutic potential of these SMIs of a critical immune checkpoint, and (iii) reaffirm the therapeutic promise of CD40-CD40L blockade in islet transplantation and T1D prevention. Thus, CD40L-targeting SMIs could ultimately lead to alternative immunomodulatory therapeutics for transplant recipients and prevention of autoimmune diseases that are safer, less immunogenic, more controllable (shorter half-lives), and more patientfriendly (i.e., suitable for oral administration, which makes them easier to administer) than corresponding antibody-based interventions.