CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells.

CD3-epsilon(CD3e) immunotoxins (IT), a promising precision reagent for various clinical conditions requiring effective depletion of T cells, often shows limited treatment efficacy for largely unknown reasons. Tissue-resident T cells that persist in peripheral tissues have been shown to play pivotal roles in local and systemic immunity, as well as transplant rejection, autoimmunity and cancers. The impact of CD3e-IT treatment on these local cells, however, remains poorly understood. Here, using a new murine testing model, we demonstrate a substantial enrichment of tissue-resident Foxp3+ Tregs following CD3e-IT treatment. Differential surface expression of CD3e among T-cell subsets appears to be a main driver of Treg enrichment in CD3e-IT treatment. The surviving Tregs in CD3e-IT-treated mice were mostly the CD3edimCD62Llo effector phenotype, but the levels of this phenotype markedly varied among different lymphoid and nonlymphoid organs. We also found notable variations in surface CD3e levels among tissue-resident T cells of different organs, and these variations drive CD3e-IT to uniquely reshape T-cell compositions in local organs. The functions of organs and anatomic locations (lymph nodes) also affected the efficacy of CD3e-IT. The multi-organ pharmacodynamics of CD3e-IT and potential treatment resistance mechanisms identified in this study may generate new opportunities to further improve this promising treatment.

Comparison of CD3e Antibody and CD3e-sZAP Immunotoxin Treatment in Mice Identifies sZAP as the Main Driver of Vascular Leakage.

Anti-CD3-epsilon (CD3e) monoclonal antibodies (mAbs) and CD3e immunotoxins (ITs) are promising targeted therapy options for various T-cell disorders. Despite significant advances in mAb and IT engineering, vascular leakage syndrome (VLS) remains a major dose-limiting toxicity for ITs and has been poorly characterized for recent "engineered" mAbs. This study undertakes a direct comparison of non-mitogenic CD3e-mAb (145-2C11 with Fc-silentTM murine IgG1: S-CD3e-mAb) and a new murine-version CD3e-IT (saporin-streptavidin (sZAP) conjugated with S-CD3e-mAb: S-CD3e-IT) and identifies their distinct toxicity profiles in mice. As expected, the two agents showed different modes of action on T cells, with S-CD3e-mAb inducing nearly complete modulation of CD3e on the cell surface, while S-CD3e-IT depleted the cells. S-CD3e-IT significantly increased the infiltration of polymorphonuclear leukocytes (PMNs) into the tissue parenchyma of the spleen and lungs, a sign of increased vascular permeability. By contrast, S-CD3e-mAbs-treated mice showed no notable signs of vascular leakage. Treatment with control ITs (sZAP conjugated with Fc-silent isotype antibodies) induced significant vascular leakage without causing T-cell deaths. These results demonstrate that the toxin portion of S-CD3e-IT, not the CD3e-binding portion (S-CD3e-mAb), is the main driver of vascular leakage, thus clarifying the molecular target for improving safety profiles in CD3e-IT therapy.