Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti–PD-1 in melanoma

Significance Regulatory T cells (Tregs) infiltrate tumors in various cancers and promote an immunosuppressive microenvironment that hinders antitumor immunity. Denileukin diftitox, a diphtheria-toxin–based fusion protein that depletes Tregs, was approved for the treatment of T cell malignancies, but its clinical use was limited due to the presence of protein aggregates and toxicity associated with vascular leakage. Here, we report the production of a second-generation IL-2 receptor-targeted, fully-folded, monomeric diphtheria fusion toxin and a V6A mutant variant that showed reduced vascular leak in vitro and reduced lethality in mice. In a mouse model of melanoma, we found a significant decrease in tumor growth associated with reduction in Tregs when the protein was tested as monotherapy or in combination with checkpoint blockade.