Abstract 11598: In Silico Identification of Immunogenic Cardiac Troponin I Epitopes and Validation in Patients Treated With Immune Checkpoint Inhibitors

Abstract

Introduction: Immune checkpoint inhibitors (ICIs) have contributed to the recent decline in death due to cancers like melanoma. However, serious cardiotoxic side effects (like fatal myocarditis) have been reported. Autoimmunity against cardiac proteins has been suspected but the underlying mechanism is not known. Hypothesis: ICI treatment induces the formation of autoantibodies against immunogenic sequences of cardiac troponin I (cTn I). Methods: We identified antigenic sequences in cardiac myosin and troponin I by bioinformatic analyses, including T- and B-cell epitope prediction methods. The sequences with the highest antigenicity scores were coated on plates for enzyme-linked immunosorbent assays (ELISAs). Sera from ICI-treated mouse models and patients were used as primary antibodies to detect self-reactivity against these peptides. Mice were treated every 3 days with a total of 17 doses of PD-1, CTLA-4, a combination of both, or isotype IgG control antibodies (Abs). For clinical samples, sera was taken from 26 ICI-treated patients with melanoma, including pre-treatment samples from 20 patients. Results: From the in silico analysis, we identified 5 cTn I and 2 myosin sequences as antigenic. Using ICI-treated mouse serum, we screened these peptides and found cross-reactivity with cTn I peptide T5, which reflects a flexible linker and portion of a helix in cTn I. After completing ICI treatment in mice, there was significant reactivity to T5 in combination treatment versus control (Absorbance: IgG 0.135±0.037, CTLA-4+PD-1 Abs 0.247±0.060, IgG vs. CTLA-4+PD-1 Abs p=0.02, n=5-9). There were also increased autoantibodies in combination treatment compared to CTLA-4 Abs (CTLA-4 Abs 0.123±0.040, CTLA-4 vs. CTLA-4+PD-1 Abs p=0.001, n=9-10). In our clinical studies, there was positive reactivity against peptide T5 of 6-7 fold change in 4 subjects (15% of patients) with late stage melanoma after the initiation of ICI therapy, as compared to the average pre-treatment value. Conclusions: Our study showed that ICI-therapy induced immune-reactive antibodies directed against antigenic targets of cTn I in both animal models and patients. These data suggest a novel mechanism of ICI-induced cardiotoxicity, which warrants further investigation.