A humanized IL-2 mutein expands Tregs and prolongs transplant survival in preclinical models
Orhan Efe, Rodrigo B. Gassen, Leela Morena, Yoshikazu Ganchiku, Ayman Al Jurdi, Isadora T. Lape, Pedro Ventura-Aguiar, Christian LeGuern, Joren C. Madsen, Zachary Shriver, Gregory J. Babcock, Thiago J. Borges, Leonardo V. Riella
Orhan Efe, Rodrigo B. Gassen, Leela Morena, Yoshikazu Ganchiku, Ayman Al Jurdi, Isadora T. Lape, Pedro Ventura-Aguiar, Christian LeGuern, Joren C. Madsen, Zachary Shriver, Gregory J. Babcock, Thiago J. Borges, Leonardo V. Riella
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Research Article Immunology

A humanized IL-2 mutein expands Tregs and prolongs transplant survival in preclinical models

Abstract

Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.

Authors

Orhan Efe, Rodrigo B. Gassen, Leela Morena, Yoshikazu Ganchiku, Ayman Al Jurdi, Isadora T. Lape, Pedro Ventura-Aguiar, Christian LeGuern, Joren C. Madsen, Zachary Shriver, Gregory J. Babcock, Thiago J. Borges, Leonardo V. Riella

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Figure 4

Effect of mIL-2 in a minor-mismatch skin transplant model.

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Effect of mIL-2 in a minor-mismatch skin transplant model. (A) Illustrat...
(A) Illustration of the experimental design and (B) Kaplan-Meier graph of the graft survival in B6.mOVA to WT B6 mouse skin transplantation (n = 10–12/group, data are representative of 3 independent experiments). (C) Flow cytometric analyses of peripheral Tregs and CD8+ T cells on days 0, 11, and 24. (D) Representative flow cytometric gating and analysis of OVA tetramer+CD4+ T cells using a combination of OVA329–337:I-Ab and OVA325–335:I-Ab tetramers and graph analyses showing the percentages of (E) Ki67+ and (F) CD44+tetramer+CD4+ Tconv cells in the DLNs on day 12. (G) Representative flow cytometric gating and analysis of the total tetramer+ Tregs and (H) percentages of Ki67+tetramer+ Tregs. (I) Analysis of the frequencies of NK cells. (J) Representative flow cytometry gating and percentages CD8+ T cells positive for the OVA257–264:H-2Kb tetramer and frequencies of (K) Ki67+ and (L) CD44+ cells in CD8+tetramer+ T cells (n = 4/group). (M) Representative images of H&E staining and IHC with antibodies against FOXP3 and CD3 of the skin grafts at day 12. Dashed line–outlined rectangular areas are shown at ×2 higher magnification on the left lower corners of the FOXP3 and CD3 IHC images. Graphs show the counts of (N) CD3+ and (O) FOXP3+ cells, (P) the ratios of FOXP3+ to CD3+ cell counts, and (Q) the counts of CD31+ vessels in 2 random high-powered fields per graft (n = 4/group, data are representative of a single experiment). Scale bars: 50 μm. Graphs show the mean ± SD and 1-way (D–Q) or 2-way (C) ANOVA with Tukey’s multiple-comparison test was used for the group comparisons. The long-rank test was used for graft survival comparisons (B). HPF, high-powered field.