Meta-Analysis of Interleukin-2 Receptor Antagonists as the Treatment for Steroid-Refractory Acute Graft-Versus-Host Disease

Acute graft-versus-host disease (aGVHD) is a major complication after allogeneic hematopoietic stem cell transplantation (HSCT). Corticosteroid is the first-line treatment for aGVHD, but its response rate is only approximately 50%. At present, no uniformly accepted treatment for steroid-refractory aGVHD (SR-aGVHD) is available. Blocking interleukin-2 receptors (IL-2Rs) on donor T cells using pharmaceutical antagonists alleviates SR-aGVHD. This meta-analysis aimed to compare the efficacy and safety of four commercially available IL-2R antagonists (IL-2RAs) in SR-aGVHD treatment. A total of 31 studies met the following inclusion criteria (1): patients of any race, any sex, and all ages (2); those diagnosed with SR-aGVHD after HSCT; and (3) those using IL-2RA-based therapy as the treatment for SR-aGVHD. The overall response rate (ORR) at any time after treatment with basiliximab and daclizumab was 0.81 [95% confidence interval (CI): 0.74–0.87)] and 0.71 (95% CI: 0.56–0.82), respectively, which was better than that of inolimomab 0.54 (95% CI: 0.39–0.68) and denileukin diftitox 0.56 (95% CI: 0.35–0.76). The complete response rate (CRR) at any time after treatment with basiliximab and daclizumab was 0.55 (95% CI: 0.42–0.68) and 0.42 (95%CI: 0.29–0.56), respectively, which was better than that of inolimomab 0.30 (95% CI: 0.16–0.51) and denileukin diftitox 0.37 (95% CI: 0.24–0.52). The ORR and CRR were better after 1-month treatment with basiliximab and daclizumab than after treatment with inolimomab and denileukin diftitox. The incidence of the infection was higher after inolimomab treatment than after treatment with the other IL-2RAs. In conclusion, the efficacy and safety of different IL-2RAs varied. The response rate of basiliximab was the highest, followed by that of daclizumab. Prospective, randomized controlled trials are needed to compare the efficacy and safety of different IL-2RAs.

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

Background: Denileukin diftitox (trade name, Ontak) is the first recombinant immunotoxin (IM), in which the binding domain of diphtheria toxin has been replaced by the amino acid sequence of human interleukin-2 (DT389IL-2) using genetic engineering. Purity, stability, and structural property of the protein are critical factors for the scale-up production of this fusion protein. In this IM, location 519 has free cysteine residue that leads to cross S-S bound formation in the refolding process and, as a result, misfolding/aggregation of the protein may occur. Materials and Methods: To inhibit misfolding/aggregation, we substituted cysteine 519 by a serine residue with site-directed mutagenesis, and then the ability of the mutated protein for binding to the IL-2 receptor was predicted and determined by bioinformatics tools. For this purpose, the sequence of the denileukin diftitox was adopted from Drugbank, and the mentioned substitution applied. Two methods determined the folding of the fusion protein: de novo modeling method (by utilizing the I-TASSER database) and homology modeling method (by using some databases and tools, including Swiss-Model, PHYRE2, M4T, ModWeb, RaptorX, and EasyModeller). Finally, the ability of the proteins for binding to the IL-2 receptor was investigated by pyDock and Zdock docking servers, as well as Hex software. Results: The result showed that the mutated form (C519S) of this protein folds appropriately, and the ΔG of the models, measured by STRUM, showed no significant variation. Also, docking analysis has shown that the protein can efficiently bind to the IL-2 receptor without any substantial changes in the binding energy. Conclusion: The present study shows that the suggested mutation of this protein can be an acceptable replacement for denileukin diftitox with a similar affinity and a more proper refolding process.

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

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin–based fusion protein that depletes CD25-positive cells including regulatory T cells and has been 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-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin usingCorynebacterium diphtheriaethat secretes fully folded, biologically active, monomeric s-DAB-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-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-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-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti–programmed cell death-1 (anti–PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-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.

A second generation IL-2 receptor-targeted diphtheria fusion toxin exhibits anti-tumor activity and synergy with anti-PD-1 in melanoma

Denileukin 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.