Abstract 2976: A single administration of DNA-encoded tri-specific T cell engager (DTriTE) targeting EGFRvIII, IL13Ra2, and CD3 shows complete tumor clearance in an orthotopic challenge model of heterogeneous GBM

Abstract Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumors in adults with a five-year survival rate less than 5%. In recent years, immunotherapeutic approaches that target a tumor-associated antigen (TAA), including EGFRvIII-targeted cancer vaccine and adoptive cell transfer, have been examined in clinical studies, which so far have shown limited efficacy in GBM patients. GBM is highly heterogeneous for targeting antigen expression. An immunotherapeutic agent that harnesses multiple TAAs simultaneously may mitigate tumor antigen escape and improve efficacy in GBM patients. Here, we describe a DNA-encoded tri-specific T cell engager (DTriTE) that targets CD3 and two TAAs, EGFRvIII and IL13Ra2, which are expressed in up to 30% and 75% of GBM cases, respectively. We designed EGFRvIII-IL13Ra2-DTriTE to include single-chain variable fragments targeting IL13Ra2, CD3, and EGFRvIII in a series, fused with glycine-serine linkers. EGFRvIII-IL13Ra2-DTriTE exhibited robust binding activities to both EGFRvIII+ and IL13Ra2+ tumor cells and primary T cells, inducing robust T cell activation and anti-tumor cytotoxicity. EGFRvIII-IL13Ra2-DTriTE showed durable in vivo expression of over 10 weeks after a single IM injection in NSG mice. We developed an orthotopic challenge of a heterogeneous GBM model, wherein a mixture of EGFRvIII+ tumor cells and IL13Ra2+ tumor cells were planted in the brains of NSG mice and primary human T cells were peripherally delivered. A single IM administration of EGFRvIII-IL13Ra2-DTriTE resulted in complete tumor clearance. An administration of DNA-encoded bispecific T cell engager (DBTE) targeting EGFRvIII alone induced tumor clearance in 3 of 10 (30%) and limited tumor control in 5 of 10 (50%) animals. Similarly, mono-targeting of IL13Ra2 alone induced tumor clearance in 1 of 9 (11%) and limited tumor control in 4 of 9 (44%) animals. EGFRvIII-IL13Ra2-DTriTE treatment exhibited significantly improved survival with 8 of 8 (100%) animals survived, while treatments of single EGFRvIII-DBTE, IL13Ra2-DBTE or a vehicle control resulted in 4 of 10 (40%), 4 of 9 (44%), and 0 of 10 (0%) animals survived, respectively. This study shows that dual tumor-targeting approach using DTriTE is highly effective at treating antigenically heterogeneous GBM tumors. Further study of DTriTE multi-antigen approaches for GBM and other heterogeneous tumors appears important. Citation Format: Daniel Hongil Park, Pratik Bhojnagarwala, Kevin Liaw, Devivasha Bordoloi, Ebony N. Gary, David B. Weiner. A single administration of DNA-encoded tri-specific T cell engager (DTriTE) targeting EGFRvIII, IL13Ra2, and CD3 shows complete tumor clearance in an orthotopic challenge model of heterogeneous GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2976.