IMMU-73. A Novel Tri-Specific T Cell Engager Improves <i>In Vivo</i> Outcomes by Addressing Glioblastoma Heterogeneity

Abstract Glioblastoma (GBM) remains a lethal brain tumor with a poor prognosis. Pronounced intratumoral heterogeneity remains a significant barrier to effective targeted immunotherapies. To address this, we developed a tri-specific T cell engager (TriTE) that targets IL13Rα2, EGFR, and the mutant variant EGFRvIII, three tumor-associated antigens frequently overexpressed in GBM. Here, we demonstrate that TriTE was designed to bind to CD3 on T cells and all three tumor antigens. In vitro, TriTE induced potent T-cell activation and cell-mediated cytotoxicity against patient-derived xenograft (PDX) GBM cells. Notably, this targeted killing was selective to GBM cells, with TriTE sparing normal EGFR-expressing cells, including primary human keratinocytes and lung fibroblasts. Pharmacokinetic analysis demonstrated that the TriTE had a serum half-life of approximately 41 hours and accumulated efficiently within intracranial tumors. In vivo, we modeled intratumoral heterogeneity through co-orthotopic injection of GBM6 and GBM39 PDX lines in humanized mice reconstituted with human T cells. In this model, TriTE significantly extended mouse survival, with 45% of treated animals achieving long-term survival, and this benefit exceeded that of BTE treatment (n= 10-11, p&amp;lt;0.0001). The efficacy of the TriTE was further validated in de novo-formed p53-/- PTEN-/- GBM tumors expressing human IL13Rα2 and EGFRvIII. These results demonstrate that TriTE effectively overcomes GBM heterogeneity, elicits robust and selective T cell-mediated cytotoxicity, and significantly prolongs survival in preclinical GBM models. These findings strongly support the clinical development of TriTE as a promising immunotherapeutic approach for GBM.