Synthetic and Immunoengineering Strategies to Modulate the Tumor Microenvironment for Enhanced Cancer Immunotherapy

Cancer immunotherapy has transformed oncology by enabling immune-mediated tumor elimination, with immune checkpoint inhibitors and adoptive cell therapies providing durable benefit in some patients. However, most solid tumors show limited responses due to resistance and low efficacy. A major barrier is the tumor microenvironment (TME), which suppresses antitumor immunity through metabolic constraints, inhibitory signaling, impaired immune cell trafficking, and T-cell dysfunction. This highlights the need for multifaceted strategies beyond immune activation alone. The overarching aim of my PhD thesis was to develop targeted approaches to overcome key TME-imposed barriers limiting cytotoxic CD8â º T-cell-mediated tumor control. I investigated four complementary strategies: First, I leveraged nutrient competition by restricting glutamine, a key amino acid for tumor growth. While glutamine inhibition using 6-Diazo-5-oxo-L-norleucine impairs tumor cells, it causes toxicity. To address this, we developed a redox-responsive, tumor-targeted prodrug with improved safety compared to JHU083 and synergy with immune checkpoint blockade. I elucidated its release mechanism, demonstrating rapid plasma activation and enzyme-dependent cleavage. Second, I enhanced CAR T-cell migration by increasing chemokine receptor expression. Since â coldâ tumors limit immune infiltration, we engineered CAR T cells to express CCR7 to improve responsiveness to CCL21 gradients. These cells showed enhanced functional responses in vitro without compromising cytotoxicity. Third, I addressed T-cell exhaustion using cytokine-based immunoengineering. While terminally exhausted T cells retain cytotoxicity, they lack proliferative capacity. Treatment with an Fcâ IL-4 fusion protein induced functional reinvigoration and antitumor activity but did not sustain proliferation. Combining IL-4 with other cytokines enhanced T-cell activity in vitro, yet in vivo results revealed context-dependent effects and dominance of IL-15 signaling rather than true synergy. Fourth, I developed a molecular probe to visualize active cytotoxic T cells by targeting granzyme B. Although the probe was efficiently activated, cellular internalization influenced the signal, complicating the interpretation of cytotoxic activity. In summary, this work demonstrates the versatility of targeted strategies to overcome TME barriers and advances understanding of immune regulation and visualization. Emphasizing mechanistic precision and context-dependent effects provides principles for developing more effective and predictable immunotherapies for solid tumors.