Treg depletion in non‐human primates using a novel diphtheria toxin‐based anti‐human CCR4 immunotoxin

Abstract

Regulatory T cells (Treg) play an important role in modulating the immune response and has attracted increasing attention in diverse fields such as cancer treatment, transplantation and autoimmune diseases. CC chemokine receptor 4 (CCR4) is expressed on the majority of Tregs, especially on effector Tregs. Recently we have developed a diphtheria-toxin based anti-human CCR4 immunotoxin for depleting CCR4+ cells in vivo. In this study, we demonstrated that the anti-human CCR4 immunotoxin bound and depleted monkey CCR4+ cells in vitro. We also demonstrated that the immunotoxin bound to the CCR4+Foxp3+ monkey Tregs in vitro. In vivo studies performed in two naive cynomolgus monkeys revealed 78–89% CCR4+Foxp3+ Treg depletion in peripheral blood lasting approximately 10 days. In lymph nodes, 89–96% CCR4+Foxp3+ Tregs were depleted. No effect was observed in other cell populations including CD8+ T cells, other CD4+ T cells, B cells and NK cells. To our knowledge, this is the first agent that effectively depleted non-human primate (NHP) Tregs. This immunotoxin has potential to deplete effector Tregs for combined cancer treatment.

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

“…1A). These results are consistent with the previous binding analysis performed with human CCR4 + CCRF-CEM leukemia cell line, human and monkey PBMC (Wang et al, 2015, 2016). In vitro depletion assays demonstrated a dose-dependent depletion of CCR4 + porcine PBMC with the foldback diabody version showing the most efficacy (Fig.…”

“…Based on our previous data (Wang et al, 2015, 2016) as well as the in vitro binding and depletion data in this study (Fig. 1A–C), foldback diabody CCR4 immunotoxin was chosen for the in vivo porcine CCR4 + Treg depletion.…”

“…Surface staining and flow cytometry analysis using porcine whole blood and BD FACS lysing solution was performed as described (Wang et al, 2016). Intracellular staining was performed to detect porcine Foxp3 as described (Wang et al, 2016).…”

“…Intracellular staining was performed to detect porcine Foxp3 as described (Wang et al, 2016). 20,000 events (lymphocytes or PBMC) for each sample were collected using BD FACSCalibur (BD Biosciences, San Jose, CA).…”

“…Recently, we have developed a novel diphtheria toxin-based anti-human CCR4 immunotoxin using unique diphtheria toxin-resistant yeast Pichia Pastoris expression system (Wang et al, 2015). In vivo efficacy for targeting human CCR4 + tumors was characterized using human CCR4 + tumor-bearing NOD/SCID IL-2 receptor γ −/− ( NSG ) mouse model (Wang et al, 2015) and in vivo efficacy for targeting CCR4 + Tregs was characterized using two naive cynomolgus monkeys (Wang et al, 2016). In the current study, we demonstrate cross-reactivity of this reagent to swine CCR4 and demonstrate its efficacy to deplete CCR4 + Foxp3 + porcine Tregs in two naive MGH miniature swine.…”