GLP1-E2 therapy delays autoimmune diabetes in late-stage prediabetic NOD mice and potentiates low-dose anti-CD3 therapy for enhanced disease protection

AIMS/HYPOTHESIS: Anti-CD3 monoclonal antibody (aCD3) delays progression to stage 3 type 1 diabetes in high-risk individuals by modulating autoimmune activity. Nevertheless, responses remain variable and transient, with therapy providing only indirect beta cell protection. We investigated whether glucagon-like peptide-1-17ß-oestradiol conjugate (GLP1-E2), a beta cell-targeted fusion compound that enhances beta cell survival and function, could potentiate a short low-dose aCD3 course in preventing autoimmune diabetes in NOD mice. We hypothesised that co-targeting immune dysregulation and beta cell fragility would provide complementary and potentially synergistic benefits, resulting in more durable protection than either monotherapy. METHODS: for 18 weeks. Mice were monitored longitudinally for diabetes onset. The pancreas was analysed by spatial transcriptomics and immunostaining to assess immune infiltration, beta cell integrity and molecular pathway alterations. RESULTS: At 30 weeks of age, diabetes incidence was 77% in untreated controls, 66% in mono aCD3-treated mice and 61% in mono GLP1-E2-treated mice. Combination therapy significantly reduced diabetes incidence to 38% (p≤0.001) and delayed disease onset by 6 weeks, with sustained protection persisting for 5 weeks after treatment cessation. GLP1-E2 monotherapy reduced islet immune cell infiltration to a similar extent as aCD3 mono- and combination therapy, without affecting peripheral lymphocyte counts. Spatial transcriptomics showed increased gene responses linked to beta cell stress (Hspa5, Eif2ak3, Xbp1, Ddit3), dedifferentiation (Cd81), 'disallowed' genes (Oat, Igfbp4), antigen presentation (H2-K1, H2-Q6, H2-Ab1, H2-Eb1) and inflammation (Cxcl10, Cxcl9, Ccl5) during disease progression. These processes were attenuated by mono- and combination therapy, with aCD3 mostly restoring beta cell identity and GLP1-E2 reducing beta cell stress and immunogenicity. Staining for CD81 and TUNEL in 17-week-old treated mice revealed levels comparable to 12-week-old normoglycaemic NOD mice, while being increased in 17-week-old untreated mice. This reduced beta cell dedifferentiation and death was associated with improved beta cell protection and better preservation of beta cell mass at 26.5 weeks compared with new-onset (diabetic) mice. CONCLUSIONS/INTERPRETATION: Low-dose aCD3 or GLP1-E2 monotherapy delayed diabetes onset and preserved beta cell mass in female NOD mice, while the combination provided substantially superior protection. Simultaneously targeting immune dysregulation and beta cell vulnerability highlights the potential of combination therapy to enhance and prolong immunotherapeutic efficacy in type 1 diabetes.