Non-Invasive Detection of Immunotherapy-induced Adverse Events

4 Objectives: Significant advances in immune-checkpoints roles in cancer have led to the development of immune-checkpoint inhibitors with strikingly positive clinical outcomes across multiple tumor types. As immune-checkpoints contribute to immune homeostasis, perturbing these pathways can facilitate the loss of immunological self-tolerance in patients. These therapy-induced immune-related adverse events (irAEs) have presented a major obstacle for safely administering checkpoint inhibitors: irAEs are frequent, with astonishing occurrence numbers (up to 91%) of patients in combination treatment, and may present as life-threatening events which require timely patient management. Early evaluation and diagnosis is critical to achieve safe resolution of irAE. However, the diagnosis of irAE can be challenging, and currently relies primarily on invasive biopsies with their attendant risks. Since the time course for developing irAEs is variable, can occur after one dose or after several months of therapy and can affect multiple organs, a non-invasive approach of detecting irAEs is urgently needed. Herein, we demonstrate the use of a granzyme B targeted peptide (GZP), radiolabeled with a PET isotope (68Ga), as an agent for irAE identification in a mouse model of irAE clinical symptoms. Furthermore, the presence of granzyme B was also evaluated in kidney samples of clinical patients that developed checkpoint-inhibitor associated nephritis. Methods: irAEs mimicking clinical symptoms were generated using the Foxp3-GFP-DTR mouse strain bearing MC38 tumors. The induction of Treg depletion in the model was through i.p. injection of diphtheria toxin (DT) followed by treatment with inhibitory antibodies targeting the immune checkpoints PD-1 and CTLA-4 as well as an agonistic antibody targeting the co-stimulatory immune-checkpoint CD-137. PBS-treated and a group followed by a 5-day administration of dexamethasone (i.p.) were also studied. PET images and biodistribution studies were collected after injection of the granzyme B specific imaging agent 68Ga-mGZP. Major organs were submitted for immunofluorescence (IF) and H&E staining as well as flow cytometry of a 10-antibody panel to assess cytotoxic CD8 and Treg activation. As a confirmatory study, IF staining detecting the presence of granzyme-B was carried out in kidney samples of clinical patients with checkpoint-inhibitor associated nephritis. Results: Signs of the development of irAEs were clearly observed in the group of mice treated with checkpoint inhibitors (dermatitis and colitis). PET/CT imaging revealed a significantly higher uptake of the probe in spleen, kidney, colon and liver of the groups treated with immune-checkpoint inhibitors (Fig.1), which decreased after immunosuppressor administration. Biodistribution studies as well as IF staining confirmed the high correlation between the presence of irAEs and granzyme B expression with affected organs, as well as the capability of the imaging agent in identifying affected organs (Fig.2). Flow cytometry revealed that Treg depletion together with anti-CD137 administration expanded intra-organ immune infiltrate (CD3) and increased activated markers of immune cells in multiple organs (Fig. 4), which diminished after administration of dexamethasone. Noteworthy, immunofluorescence staining of kidney samples of four cancer patients with irAEs clearly demonstrated the presence of granzyme B in all samples tested. Conclusions: Altogether, our results demonstrate the ability of a non-invasive agent (radiolabeled GZP) in detecting immune-related adverse events, which is, to the best of our knowledge, unprecedented. A complex range of studies (IF, Flow Cytometry, IHC, Biodistribution) was performed. More importantly, granzyme B presence was observed in kidney samples of clinical cancer patients that developed checkpoint-inhibitor associated nephritis, warranting the possibility of a direct translation between preclinical and clinical settings.