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

Abstract

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.

CD3e-immunotoxin spares CD62LloTregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehiT cells

“…(61) We have recently developed a new murine-version CD3e-IT (S-CD3e-IT) whose safety and treatment efficacy profile is comparable to those of the "second-generation" recombinant CD3e-ITs. (46) Here, we demonstrate the significant enrichment of Forkhead box transcription factor (Foxp3) Tregs in the murine model following CD3e-IT. We used the new murine testing model to systemically characterize Foxp3+ Treg enrichment in multiple organsincluding the peripheral blood, spleen, lung, Peyer's Patches, 5 types of lymph nodes (mesenteric, inguinal, mandibular, mediastinal, and lumbar), thymus, and bone marrowusing intravascular staining techniques to analyze circulating and tissue-resident cells separately.…”

“…Male C57BL/6J mice were injected into retro-orbital sinus with 15 g S-CD3e-IT in sterile 200 l PBS twice a day for four consecutive days and were euthanized on day 6 as previously described (46). On the day of euthanasia, mice were injected into the retro-orbital sinus with a total of 3 mg of PE/Cy7-CD45.2 (104,BioLegend) in 200 l sterile DPBS.…”

“…All samples were acquired on a Cytek Aurora, and the data were analyzed with FlowJo software (BD Bioscience). The absolute number of leukocytes was calculated using CountBright TM as previously described (46).…”

“…( 44 IT treatments in a few selected organs. (35,46) Most past studies have focused almost exclusively on total peripheral blood, lymph node and bone marrow cells in animal models (34,37,(58)(59)(60) and total peripheral blood in humans,(1) without drawing any clear distinction between circulating and tissue-resident populations.…”

“…Only a few recent studies, including our own, have shown differential depletion efficiencies for tissue-resident T cells by anti-T-cell mAb and CD3e-IT treatments in a few selected organs. (35, 46) Most past studies have focused almost exclusively on total peripheral blood, lymph node and bone marrow cells in animal models(34, 37, 58–60) and total peripheral blood in humans,(1) without drawing any clear distinction between circulating and tissue-resident populations.…”