Development of stimuli-responsive chemistry for tumor-targeted immunotherapy

Cancer immunotherapy is the science of boosting the immune response of patients to fight cancer. Recent advances have brought up treatments with huge success in the clinic. Patients with ad-vanced-stage cancers were long-term cured through checkpoint inhibitors or CAR-T cell administration. However, only a subset of patients could benefit from these new therapies, despite the high potential of the underlying technologies. This is largely due to the high variability of cancers among patients and the development of therapy resistance, problems which can only be solved by finding adaptable and targeted immunotherapies, which can be combined and personalized to patients. In this PhD thesis, I used chemistry-based approaches to develop new formulations for im-munoenhancing small molecules and proteins. The concept of stimuli-responsive chemical linkers ac-companied me throughout my projects and set the base for my investigations towards improving the safety and specificity of cancer immunotherapy treatments.Enhancing tumor infiltration of T cells using cytokine nanogels composed of interleukin-15-superagonist (IL15-SA) proteins and radical-oxygen-species (ROS)-responsive crosslinkers. IL15-SA has previously been explored as a powerful lifetime-extended cytokine to boost immune responses in patients. However, adverse effects arising with systemic administration limit its use in high doses. I developed tumor-specific nanosized hydrogels (nanogels) which were designed to deliver the IL15-SA cytokines preferentially to the tumor site and thereby would reduce off-site adverse effects. I used bo-ronic-ester-based ROS-responsive chemical linkers which crosslinked the IL15-SA cytokines for tu-mor-targeted delivery. The linkers were designed to tracelessly release the IL15-SA cytokines to the tumor microenvironment (TME) upon reaction with ROS, thereby returning their potential locally and over a prolonged time. Potentiating immune checkpoint blockade (ICB) with a redox-responsive prodrug of the glutamine inhibitor 6-Diazo-5-oxo-L-norleucine (DON). Glutamine metabolism is a known vulnerability in tu-mors. Clinical trials with the small molecule DON showed potency, but severe toxicity, in particular in the gastrointestinal (GI) tract. The formulation of DON into protease-responsive prodrugs has previ-ously been demonstrated to decrease adverse effects and the treatment synergized with ICB in different mouse models of cancer. Here, I synthesized and applied a novel redox-responsive DON prodrug, which showed equivalent efficacy, but improved safety compared to the state-of-the-art DON prodrug JHU083 in vivo. Moreover, I demonstrated synergy with ICB and identified the main immune cell me-diators. In summary, my research provides novel strategies using stimuli-responsive chemical linkers for the application in cancer immunotherapy. These projects illustrate the versatility and potential of chemical formulations for the delivery of immunoenhancing proteins and small molecules. Further-more, I contributed to the understanding of the mechanism of combining metabolic drugs with check-point inhibitors. The concept of traceless release of immunomodulatory agents as elaborated in this thesis, will potentially find novel applications across diverse projects in cancer immunotherapy.