Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells

Peraino, Jaclyn Stromp; Schenk, Marian; Li, Guoying; Zhang, Huiping; Farkash, Evan A.; Sachs, David H.; Huang, Christene A.; Duran‐Struuck, Raimon; Wang, Zhirui

doi:10.1016/j.jim.2013.09.006

Cited by 15 publications

(21 citation statements)

References 13 publications

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“…Of note, pig islets are likely resistant to direct DT toxicity, as (1) the pig islets in DT-treated recipients survived for over 10 days after the last dose of DT (Fig. 5B); and (2) published literature support that pig cells are resistant to DT unless DT is guided to the targeted cells by ligand-receptor binding via DT fusion proteins (37). Collectively, these data suggests that Treg play a critical role in promoting and maintaining pig islet xenograft survival seen in recipients treated with the triple therapy.…”

Section: Resultsmentioning

confidence: 96%

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice

Kang

Wang

Dangi³

et al. 2017

Transplantation

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Background Xenogeneic islet transplantation is an emerging therapeutic option for diabetic patients. However, immunological tolerance to xenogeneic islets remains a challenge. Methods The current study used a pig-to-mouse discordant xenogeneic islet transplant model to examine anti-donor xenogeneic immune responses during early and late rejection, and to determine experimental therapeutic interventions that promote durable pig islet xenograft survival. Results We found that during early acute rejection of pig islet xenografts, the rejecting hosts exhibited a heavy graft infiltration with B220+ B cells and a robust anti-pig antibody production. In addition, early donor-stimulated IL-17 production, but not IFN-γ production, dominated during early acute rejection. Recipient treatment with donor apoptotic 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide-treated splenocytes (ECDI-SP) significantly inhibited anti-donor IL-17 response, and when combined with B cell depletion and a short course of rapamycin led to survival of pig islet xenografts beyond 100 days in ~65% recipients. Interestingly, treated recipients in this model experienced late rejection between 100 – 200 days posttransplant, which coincided with B cell reconstitution and an ensuing emergence of a robust anti-donor IFN-γ, but not IL-17, response. Conclusions These findings reveal that early and late rejection of pig islet xenografts may be dominated by different immune responses, and that maintenance of long-term xenogeneic tolerance will require strategies that target the temporal sequence of anti-xenogeneic immune responses.

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Section: Resultsmentioning

confidence: 96%

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice

Kang

Wang

Dangi³

et al. 2017

Transplantation

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“…Based on our experience in developing porcine IL-2 fusion toxins (Peraino et al, 2013b), we hypothesized that the bivalent human IL-2 fusion toxin would prove to be a more potent in vivo depletion agent of human CD25 + cells than the clinically-used monovalent human IL-2 fusion toxin (denileukin diftitox, Ontak®). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Western blot analysis, binding affinity and specificity analysis by flow cytometry and K d determination were all performed as previously described (Peraino et al, 2013a) using a human CD25 + T-cell lymphoma cell line HUT 102/6TG (Williams et al, 1990) (kindly provided by Dr. Robert Harrison, Anjin Group, Inc., Boston, MA). Isolation of human PBMC as well as in vitro binding analysis of the human IL-2 fusion toxin to human CD25 on PBMC using flow cytometry was performed as previously described (Peraino et al, 2013b). DT390 and human IL-2 were used as controls for all in vitro functional analysis.…”

Section: Methodsmentioning

confidence: 99%

Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells

Peraino

Zhang

Rajasekera

et al. 2014

Journal of Immunological Methods

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Regulatory T cells (Treg) constitute a major inhibitory cell population which suppresses immune responses. Thus, Treg have proven to be key players in the induction of transplantation tolerance, protection from autoimmune disease and prevention of the development of effective anti-tumor immune reactions. Treg express high levels of the high affinity interleukin-2 receptor (IL-2R) consisting of IL-2Rα (CD25) together with IL-2Rβ (CD122) and the common γ-chain (CD132). An effective reagent capable of depleting Treg in vivo would facilitate better cancer treatment and allow mechanistic studies of the role of Treg in transplantation tolerance and the development of autoimmune disease. In this study, we have developed a novel bivalent human IL-2 fusion toxin along with an Ontak®-like monovalent human IL-2 fusion toxin and compared the functional ability of these reagents in vitro. Here we show that genetically linking two human IL-2 domains in tandem, thereby generating a bivalent fusion toxin, results in significantly improved capacity in targeting human CD25+ cells in vitro. Binding analysis by flow cytometry showed that the bivalent human IL-2 fusion toxin has notably increased affinity for human CD25+ cells. In vitro functional analysis demonstrated that the bivalent isoform has an increased potency of approximately 2 logs in inhibiting cellular proliferation and protein synthesis in human CD25+ cells compared to the monovalent human IL-2 fusion toxin. Additionally, we performed two inhibition assays in order to verify that the fusion toxins target the cells specifically through binding of the human IL-2 domain of the fusion toxin to the human IL-2 receptor on the cell surface. These results demonstrated that 1) both monovalent and bivalent human IL-2 fusion toxins are capable of blocking the binding of biotinylated human IL-2 to human CD25 by flow cytometry; and 2) human IL-2 blocked the fusion toxins from inhibiting protein synthesis and cellular proliferation in vitro, thus confirming that the human IL-2 fusion toxins target the cells specifically through binding to the human IL-2 receptor. We believe that the bivalent human IL-2 fusion toxin will be a more potent, and therefore, more optimal agent than the current clinically-used monovalent fusion toxin (denileukin diftitox, Ontak®) for in vivo depletion of Treg.

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“…Human IL-2 fusion toxin was injected IP daily for 10 consecutive days starting on day 0 of tumor injection. The injected animals were observed daily for signs and symptoms of illness and scored at least twice weekly based on the parameters as previously reported by our lab (Peraino et al, 2013b). To assess the non-specific toxicity of the human IL-2 fusion toxin, two mice (n=2) injected with the bivalent human IL-2 fusion toxin alone at 100 μg/kg were also included as controls.…”

Section: Methodsmentioning

confidence: 99%

Ontak-like human IL-2 fusion toxin

Wang

Zheng

Zhang

et al. 2017

Journal of Immunological Methods

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Ontak® is a FDA-approved diphtheria toxin-based recombinant fusion toxin for treatment of human CD25+ cutaneous T cell lymphoma (CTCL). However, it has been discontinued clinically due to the production issue related to the bacterial expression system with difficult purification. Recently we have developed monovalent and bivalent human IL-2 fusion toxins targeting human CD25+ cells using advanced unique diphtheria toxin resistant yeast Pichia Pastoris expression system. In vitro efficacy characterization using human CD25+ HUT102/6TG cells demonstrated that both monovalent and bivalent isoforms are potent and the bivalent isoform is approximately two logs more potent than the monovalent isoform. In this study, we further assessed the in vivo efficacy of the human IL-2 fusion toxins using human CD25+ HUT102/6TG tumor-bearing NSG mouse model. The data demonstrated that both monovalent and bivalent human IL-2 fusion toxins significantly prolonged the survival of the human CD25+ tumor-bearing NSG mice in a dose-dependent manner. Then we further assessed the residual tumor cells from the HUT102/6TG tumor-bearing NSG mice using the residual tumor cell bearing NSG mouse model. The results demonstrated that the residual tumor cells were still sensitive to the continual treatment with the human IL-2 fusion toxin. This yeast-expressed human IL-2 fusion toxin will be a promising candidate to replace the clinically discontinued Ontak®.

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Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells

Cited by 15 publications

References 13 publications

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice

Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells

Ontak-like human IL-2 fusion toxin

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