Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: Mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15

Peraino, Jaclyn Stromp; Hermanrud, Christina; Springett, Lauren; Zhang, Huiping; Li, Guoying; Srinivasan, Srimathi; Gusha, Ashley; Sachs, David H.; Huang, Christene A.; Wang, Zhirui

doi:10.1016/j.cellimm.2012.05.004

Cited by 9 publications

(14 citation statements)

References 17 publications

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“…As shown in Figure 8, both NonGly and Bi-NonGly fusion toxins bound to the porcine PBMC in a dose dependent manner with the Bi-NonGly isoform binding stronger than its monovalent counterpart. A biotinylated anti-porcine CD25 mAb (clone# 231-3B2) was included as positive control and biotinylated porcine CD3 εγ (Peraino et al, 2012b) as a control for any background as a result of protein biotinylation.…”

Section: Resultsmentioning

confidence: 99%

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

Peraino

Schenk

et al. 2013

Journal of Immunological Methods

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Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25+ B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins’ ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25+ cells for studying immune regulation.

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Section: Resultsmentioning

confidence: 99%

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

Peraino

Schenk

et al. 2013

Journal of Immunological Methods

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“…Porcine whole blood with only the secondary staining (PE-conjugated streptavidin) served as the negative control and PE-mouse anti-human/rat CCR4 mAb (clone# 205410) as the CCR4 positive control, PE-mouse IgG2B (clone# MPC-11) for the isotype control of PE-mouse anti-human/rat CCR4 mAb (clone# 205410), PerCp-Cy5.5-mouse anti-pig CD4a mAb (clone#74-12-4) as CD4 positive control. Biotin-labeled porcine CD3-εγ (Peraino et al, 2012) was included as a negative control for background due to protein biotinylation. The stained samples were analyzed using BD FACSCalibur (BD Biosciences, San Jose, CA).…”

Section: Methodsmentioning

confidence: 99%

“…FITC-rat anti-mouse Foxp3 mAb (clone# FJK-16s) was used for the Foxp3 positive control, FITC-rat-IgG2a, κ (clone# eBR2a) for the isotype control of FITC-rat anti-mouse Foxp3 mAb (clone# FJK-16s). Biotin-labeled porcine CD3-εγ (Peraino et al, 2012) was included as a negative control for background due to protein biotinylation. The stained samples were analyzed using BD FACSCalibur (BD Biosciences, San Jose, CA).…”

Section: Methodsmentioning

confidence: 99%

Porcine Treg depletion with a novel diphtheria toxin-based anti-human CCR4 immunotoxin

Wang

Navarro-Alvarez

Shah

et al. 2016

Veterinary Immunology and Immunopathology

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Regulatory T cells (Tregs) are known to play an important role in immunoregulation and have been shown to facilitate induction of transplantation tolerance. Chemokine (C-C motif) receptor 4 (CCR4) is expressed on the surface of effector Tregs involved in controlling alloimmune and autoimmune responses. Recently we have developed a novel diphtheria-toxin based anti-human CCR4 immunotoxin for depleting CCR4+ cells in vivo. In this study, we have demonstrated that the anti-human CCR4 immunotoxin bound to porcine lymphocytes including CD4+FoxP3+ Tregs. Anti-human CCR4 immunotoxin effectively depleted CCR4+ Foxp3+ porcine Tregs in vivo. We observed depletion of up to 70–85% of the CCR4+Foxp3+ porcine Tregs in the peripheral blood and 85–91% in the lymph nodes following the anti-human CCR4 immunotoxin treatment in Massachusetts General Hospital (MGH) miniature swine. The depletion lasted for about one week with no significant reduction observed within CCR4− cell populations including CD8α+ T cells, CCR4−CD4+ T cells and B cells. In summary, anti-human CCR4 immunotoxin effectively depleted CCR4+Foxp3+ porcine Tregs in both peripheral blood and lymph nodes.

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“…Cells with only the secondary staining (PE-conjugated streptavidin) served as the negative control and PE-conjugated mouse anti-human CD25 mAb (clone# M-A251, BD Pharmingen, Cat# 555432) was used for the positive control. Biotin-labeled porcine CD3-εγ (Peraino et al, 2012b) was included as a negative control for background due to protein biotinylation. The data are representative of multiple individual experiments.…”

Section: Figmentioning

confidence: 99%

“…The Y axis indicates the biotinylated bivalent human IL-2 fusion toxin as the primary stain and the PE-conjugated streptavidin as the secondary stain. The first panel (labeled as “0 nM” of the biotinylated bivalent human IL-2 fusion toxin) is the negative control stained with the biotinylated porcine CD3 εγ (20 nM) (Peraino et al, 2012b) for any background as a result of protein biotinylation. Data are representative of two individual assays.…”

Section: Figmentioning

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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Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: Mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15

Cited by 9 publications

References 17 publications

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

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

Porcine Treg depletion with a novel diphtheria toxin-based anti-human CCR4 immunotoxin

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

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