Development of ST2 Nanobodies for Colorectal Cancer Immunotherapy - Colorectal cancer (CRC) is the third most common cancer worldwide, affecting both women and men. Advances in screening and treatment methods have increased the survival rate significantly, especially when targeting early stages of the disease. Unfortunately, once metastasis occurs, the therapeutic success rate significantly drops. A breakthrough treatment for advanced disease are the so-called immune checkpoints, particularly anti-programmed death 1. Yet, this immunotherapy is most effective in patients with microsatellite instability tumors, a small minority of the patients. However, driven by the success of existing immune checkpoints, there is the need to identify other immune checkpoint targets and generate much-needed treatments for other patients. We previously discovered ST2 (interleukin-33 receptor, stimulation 2), a novel and promising immune checkpoint target. Excited by the potential of ST2, we propose to develop anti-ST2 theranostic single domain antibodies (sdAbs, or nanobodies; Nbs) for both diagnostic and therapeutic purpose. To address this need, we will generate and characterize murine and human anti-ST2 Nbs, while also considering cross-reactive anti-ST2 Nbs. The functionality in binding and blocking for these Nbs will be determined. Non-blocking Nbs will be used for diagnostic purposes upon labeling the Nbs with Technetium-99m (⁹⁹mTc). Using this approach, we will assess the top targets with radiotracers for non-invasive single-photon emission computerized tomography (SPECT)/computed tomography (CT) imaging. This Nb screening method will also be used for ex vivo biodistribution studies, in which the accumulation of radioactivity in dissected tissues will be assessed, followed by confirmation of target (ST2) expression using flow cytometry. Using our top target Nbs, we will determine their capacity to monitor dynamic changes in the IL33/ST2 pathway in the tumor microenvironment. Since the tumor microenvironment evolves over time and could be altered by other standard-of-care treatment modalities. Next, we will uncover the essential role of the IL33/ST2 pathway in cancer metastasis using positron emission tomography (PET) imaging. Finally, we will take our anti-ST2 Nbs and assess their therapeutic potential, evaluating anti-ST2 blocking Nbs. For this, we will encode anti-ST2 Nbs in the form of mRNA and deliver it intratumorally, using endoscopy-guided injections, mimicking clinical translation. To increase the presence of anti-ST2 Nbs in the tumor microenvironment, we will engineer our mRNA to increase the local bioavailability of our anti-ST2 Nbs. Taken together, this proposal will uncover the theranostic potential of anti-ST2 Nbs, while also establishing a platform for other immune checkpoint targets for cancer immunotherapy and immunomonitoring. Animal model justification: Essential biodistribution studies and tumor growth control experiments will be carried out in advanced preclinical animal models that are critical to clinical translation of our findings.
