Tri-Specific T-Cell Engagers for Glioblastoma: A Multivalent Immunotherapy Targeting Tumor Heterogeneity

Glioblastoma multiforme (GBM) is one of the most aggressive and lethal forms of brain cancer, with a five-year survival rate of less than 5%. Despite advances in surgery, radiation, and chemotherapy, the prognosis remains grim, with most patients surviving just 12–15 months after diagnosis. GBM’s resilience to conventional treatments stems from its unique and complex biology, particularly its antigenic heterogeneity. Unlike tumors with a relatively uniform expression of antigens, GBM tumors exhibit significant variability in their molecular makeup, allowing them to evade therapies that target a single tumor-associated antigen (TAA). This diversity fuels resistance, tumor recurrence, and ultimately poor patient outcomes. One of the major challenges in treating GBM lies in its high level of intratumoral heterogeneity. Individual tumors express a range of TAAs at varying levels, with some regions of the tumor entirely lacking the targets for a given therapy. Immunotherapeutic strategies, such as chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers (BTEs), have shown promise in targeting specific antigens, but their efficacy is often limited by GBM’s ability to downregulate or lose these antigens during treatment. For example, while targeting epidermal growth factor receptor variant III (EGFRvIII) has demonstrated some success, the antigen’s patchy and inconsistent expression across tumors limits the durability of the response. Similarly, interleukin-13 receptor alpha 2 (IL-13Rα2), another prominent GBM antigen, is only present in a subset of tumor cells, further complicating treatment efforts. Recognizing these challenges, new research study published in Journal for ImmunoTherapy of Cancer  and conducted by Dr. Daniel Park, Dr. Prati Bhojnagarwala, Dr. Kevin Liaw, Dr. Devivasha Bordoloi, Dr. Nicholas Tursi, Shushu Zhao,  Dr. Zev  Binder, Professor Donald O’Rourke, Professor  David Weiner from University of Pennsylvania and the Wister Institute developed a more effective therapeutic strategy capable of overcoming GBM’s antigenic complexity. Their approach focused on designing DNA-encoded tri-specific T-cell engagers (DTriTEs), a novel class of immunotherapeutics capable of targeting multiple antigens simultaneously. By incorporating binding domains for both EGFRvIII and IL-13Rα2, along with the CD3 receptor on T cells, these tri-specific constructs aim to engage the immune system more comprehensively and minimize the likelihood of tumor escape due to antigen loss.

The research team evaluated the efficacy of their novel  DTriTEs in overcoming the challenges posed by the antigenic heterogeneity of GBM. Their first step involved designing three DTriTE constructs, each engineered to target two key tumor antigens, EGFRvIII and IL-13Rα2, while simultaneously engaging T cells via the CD3 receptor. These constructs were tested in vitro to assess their binding efficiency, cytotoxic potential, and activation of immune cells. Among the three, DT2035 emerged as the lead candidate, demonstrating robust and selective binding to cells expressing EGFRvIII and IL-13Rα2, while efficiently engaging T cells to mediate targeted killing. The cytotoxicity of DT2035 was further validated in tumor-killing assays using GBM cell lines with heterogeneous expression of the target antigens. The results showed that DT2035 effectively eliminated tumor cells, including those with mixed or low antigen expression, highlighting its versatility. Notably, DT2035 induced significant T-cell activation, characterized by the production of key antitumor cytokines such as IFN-γ, TNF-α, and IL-2. These findings were critical in demonstrating that the construct could engage immune cells to mount a potent and specific antitumor response. To assess the therapeutic potential in a more clinically relevant context, the authors conducted in vivo experiments using mouse models implanted with GBM tumors exhibiting heterogeneous antigen profiles. When administered to these mice, DT2035 produced remarkable outcomes, including significant tumor regression and prolonged survival. In one of the most rigorous tests, an intracranial GBM model mimicking the challenges of human tumors, DT2035-treated mice achieved a 67% survival rate over 120 days, far surpassing the outcomes of controls or therapies targeting a single antigen. Importantly, DT2035 maintained its activity over extended periods, highlighting its potential for durable tumor control. Beyond tumor eradication, the researchers explored how DT2035 modulates the immune system. RNA sequencing of T cells exposed to the construct revealed enhanced expression of genes associated with cytotoxicity, proliferation, and immune regulation. This included upregulation of granzyme B and IFN-γ, alongside co-stimulatory molecules that amplify T-cell activation. These insights underscored the multifaceted mechanism of DT2035, which not only targets tumor cells but also strengthens the immune response. Further tests involved patient-derived immune cells to evaluate the translational potential of the therapy. When DT2035 was introduced to peripheral blood mononuclear cells (PBMCs) from GBM patients, including those who had undergone extensive prior treatments, the results were promising. The construct consistently activated patient immune cells to produce cytokines and engage in tumor cell killing, even in cases where immune function might be compromised. These findings emphasized the potential applicability of DT2035 in real-world clinical settings.

In conclusion, the study by Professor  David Weiner  and colleagues is an advancement in addressing the therapeutic challenges of glioblastoma multiforme (GBM), a highly aggressive brain cancer with poor survival outcomes. By developing and evaluating  DTriTEs, the research introduces a novel approach to overcome the limitations of single-antigen targeting therapies. The lead construct, DT2035, demonstrates exceptional efficacy in preclinical models, offering a potent strategy to tackle GBM’s hallmark antigenic heterogeneity. The ability of DT2035 to simultaneously target two distinct tumor antigens, EGFRvIII and IL-13Rα2, while engaging T cells through CD3, sets it apart from existing immunotherapies. This multivalent design ensures that tumor cells with varying antigen profiles can be effectively targeted, reducing the risk of immune evasion—a significant limitation of current treatments. By leveraging the immune system’s cytotoxic potential, DT2035 enhances antitumor responses while maintaining specificity, minimizing the risk of off-target effects. The implications of this study extend beyond GBM treatment. The platform for designing tri-specific T-cell engagers can be adapted to other cancers characterized by antigenic heterogeneity. This adaptability opens new avenues for treating a variety of tumors that have eluded traditional single-antigen targeting approaches, marking a paradigm shift in cancer immunotherapy. Another critical aspect of this research lies in its translational potential. The findings demonstrate that DT2035 not only activates immune cells in laboratory models but also engages immune cells from GBM patients, including those with compromised immunity due to prior treatments. This underscores its potential for clinical application and suggests that DT2035 could become a vital component of combination therapies, potentially used alongside checkpoint inhibitors or other immune-modulating agents. The study also highlights the durability of the therapeutic effect, with DT2035 showing sustained antitumor activity over extended periods in preclinical models. This extended efficacy offers hope for long-term tumor control, addressing the recurrent nature of GBM. Furthermore, the use of DNA-encoded therapy provides a scalable and potentially cost-effective delivery method, paving the way for broader accessibility in clinical settings.