AbstractDenileukin diftitox (DAB1-389-IL-2, Ontak®) is a diphtheria toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells (Tregs) and was approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB1-386-IL-2(V6A), which elicits 50-fold less HUVEC monolayer permeation and is 3.7-fold less lethal to mice by LD50analysis than s-DAB1-386-IL-2 Additionally, to overcome aggregation problems, we developed a novel production method for the fusion toxin usingCorynebacterium diphtheriaethat secretes fully-folded, biologically active, monomeric s-DAB1-386-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB1-386-IL-2(V6A) and s-DAB1-386-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB1-386-IL-2(V6A) could provide a superior activity window. In a sequential dual therapy study in tumors that have progressed for 10 days both s-DAB1-386-IL-2(V6A) and s-DAB1-386-IL-2 given prior to checkpoint inhibition with anti-PD-1 antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB1-386-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.Significance StatementRegulatory T cells (Tregs) infiltrate tumors in various cancers and promote an immunosuppressive microenvironment that hinders anti-tumor 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 which showed reduced vascular leak in vitro and reduced lethality in mice. In a mouse model of melanoma, we found significant decrease in tumor growth associated with reduction in Tregs when the protein was tested as monotherapy or in combination with checkpoint blockade.
Inability of a Fusion Protein of IL-2 and Diphtheria Toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to Eliminate Regulatory T Lymphocytes in Patients With Melanoma