Glioblastoma Multiforme (GBM) is a highly aggressive brain cancer characterized by significant antigenic heterogeneity, complicating treatment and leading to poor patient outcomes. Recent efforts with monovalent therapies for GBM showed limited efficacy due to tumor escape mechanisms driven by this heterogeneity. This dissertation addresses the need for innovative immunotherapeutic strategies that can effectively target the diverse antigenic landscape of GBM and engage a broad range of immune effector populations to enhance therapeutic efficacy.To investigate this problem, two novel immunotherapeutic approaches were developed and characterized: DNA-encoded Bispecific T Cell Engagers (DBTEs) and Tri-specific T Cell Engagers (DTriTEs). DBTEs targeting EGFRvIII and HER2 were designed to simultaneously target these antigens, while DTriTEs were engineered to target EGFRvIII and IL13Rα2, engaging multiple immune cell types, including CD4+ T cells and Natural Killer T (NKT) cells. A heterogeneous GBM model was specifically developed to examine the effectiveness of these multivalent approaches in preclinical settings. The studies included tumor-killing assays, flow cytometry for immune activation, and in vivo models to assess tumor control and survival outcomes. The results demonstrated that both DBTEs and DTriTEs effectively induced potent T cell-mediated cytotoxicity across various GBM cell lines, including those resistant to standard therapies. The combination of DBTEs targeting different antigens showed enhanced tumor control and improved survival in the heterogeneous GBM model. Notably, DTriTEs outperformed the combined DBTEs, demonstrating superior efficacy in targeting multiple antigens simultaneously, activating a broader range of immune effector populations, and inducing robust and sustained anti-tumor responses. Furthermore, DTriTEs successfully induced anti-tumor cytotoxicity in patient-derived immune cells, including those from patients who had undergone prior chemoradiotherapy, underscoring their translational potential. Additionally, DTriTEs showed potential for synergistic use with other immunotherapies, particularly immune checkpoint inhibitors. In conclusion, this dissertation presents important advancements in the development of multivalent immunotherapies for GBM. By addressing the limitations of monovalent therapies, these novel DBTEs and DTriTEs offer a promising approach to overcoming GBM’s antigenic heterogeneity and improving patient outcomes. The findings provide a strong foundation for further development of these therapies, potentially leading to more effective and durable treatments for this challenging cancer.