Post-Translational Regulation of 4-1BB, an Emerging Target for Cancer Immunotherapy

Cancer is well known as a disease involving genetic disorders, which make them distinguishable from normal tissue by the altered molecular signatures. Theoretically, malignant cells can be recognized and attacked by innate and adaptive immune system as “non-self” species, and the idea to take advantage of host immunity to treat cancer has been discussed for over a century. Through the multi-disciplinary research efforts from immunology, cancer biology, cell engineering etc., cancer immunotherapy has been successfully translated from benchside to bedside. While the clinical application of immunotherapeutic regimens has achieved extraordinary success including the unprecedented long-term survival of metastatic melanoma patients, we must take it seriously that only a small proportion (about 20% on average) of patients benefit from immunotherapy, and many develop secondary progression after the initial response. Advancements have been made in biomarker development to identify the group the patients who may benefit from immunotherapy, yet the accuracy and adaptability remain to be improved. In general, the performance of immunotherapy is hardly satisfactory as the current situation. The effect out of T cell-mediated immune response is mediated by plenty pairs of receptorligand interactions in the immune synapse between T cells and target cells. Despite the T cell receptor-mediated first signal and CD28-mediated second signal, a huge collection of co-signals molecules serves unneglectable roles to keep the T-cell immune response fine-tuned under appropriate threshold. Inadequate co-signaling transduction result in with immune deficiency or autoimmunity depending on the type of signal (stimulatory or inhibitory). 4-1BB is a significant co-receptor which is mainly expressed on T cells and delivers activation signal to drive T cell proliferation and cytotoxicity. 4-1BB is targetable for cancer treatment and can be used as a tumorreactive T cell marker as well. Hence, it is of substantial importance to understand how cosignaling molecules, such as 4-1BB, are regulated under specific physiological or pathological conditions. Proteins are regulated at multiple levels, including transcription, translation, localization, and interaction with other biomolecules (covalently or non-covalently). Post-translational modification (PTM) constitutes a critical type of mechanism that elicit multidimensional effects to the biophysical properties of target proteins. Herein, I sought to elaborate how 4-1BB, an TNFRSF family co-stimulatory receptor, is possessed and regulated by PTMs, particularly ubiquitination and N-glycosylation. In the first part of this study, I confirmed that 4-1BB is degraded through ubiquitination-proteasome pathway and identified FBXL20 to be the E3 ligase subunit mediating 4-1BB polyubiquitination. While conducting the first section, I noticed that 4-1BB is heavily Nglycosylated and thereby dissected the biological significance of this modification which made up the second part of this study. I experimentally characterized that 4-1BB necessitates its N-glycans to be efficaciously transported to cell membrane through the secretory pathway. Plus, the glycosylated 4-1BB has short half-life. Without the spatial hindrance established by N-linked carbohydrate moieties, the exposed C121 residue of 4-1BB can be used to forms stable multimer which intracellular retention and stabilization of 4-1BB. This study uncovered the post-translational mechanisms of action of 4-1BB regulation for the first time. More fundamentally, we provided a blueprint to study the post-translational regulation network of immune receptors which may be applied for future investigations in other targets. Our ultimate hope is to be able to grasp the dynamic of key immune regulators in the context of microenvironment and based on which pair the right therapeutics with the correct populations