A Novel Therapeutic Approach using CXCR3 Blockade to Treat Immune Checkpoint Inhibitor-mediated Myocarditis

Background Immune checkpoint inhibitors (ICIs) are successful in treating many cancers but may cause immune-related adverse events. ICI-mediated myocarditis has a high fatality rate of up to 40%, with severe cardiovascular consequences. Targeted therapies for ICI myocarditis are currently lacking. Methods We used a genetic mouse model of PD-1 deletion ( MRL/Pdcd1-/- ) along with a novel drug-treated ICI myocarditis mouse model to recapitulate the disease phenotype. We performed single-cell RNA-sequencing (scRNAseq), single-cell T-cell receptor sequencing (scTCR-seq), and cellular indexing of transcriptomes and epitopes (CITE-seq) on immune cells isolated from MRL and MRL/Pdcd1-/- mice at serial timepoints. We assessed the impact of macrophage deletion in MRL/Pdcd1-/- mice, then inhibited CXC chemokine receptor 3 (CXCR3) in ICI-treated mice to assess therapeutic effect on myocarditis phenotype. Furthermore, we delineated functional effects of CXCR3 blockade on T-cell and macrophage interactions in a transwell assay. We then correlated the results in human single-cell multi-omics data from blood and heart biopsy data from patients with ICI myocarditis. Results Single-cell multi-omics demonstrated expansion of CXCL9/10+CCR2+ macrophages and CXCR3hi CD8+ effector T-lymphocytes in the hearts of MRL/Pdcd1-/- mice correlating with onset of myocarditis development. Both depletion of CXCL9/10+CCR2+ macrophages and CXCR3 blockade respectively led to decreased CXCR3hiCD8+ T-cell infiltration into the heart and significantly improved survival. A transwell assay showed that selective blockade of CXCR3 and its ligand, CXCL10 decreased CD8+ T-cell migration towards macrophages, implicating this interaction in T-cell cardiotropism towards cardiac macrophages. Cardiac biopsies from patients with confirmed ICI myocarditis demonstrated infiltrating CXCR3+ lymphocytes and CXCL9+/CXCL10+ macrophages. Both mouse cardiac immune cells and patient peripheral blood immune cells revealed expanded TCRs correlating with CXCR3hi CD8+ T-cells in ICI myocarditis samples. Conclusions These findings bring forth the CXCR3-CXCL9/10 axis as an attractive therapeutic target for ICI myocarditis treatment, and more broadly, as a druggable pathway in cardiac inflammation. ### Competing Interest Statement Dr. Wakelee is an advisory board participant of IOBiotech and Mirati, and she does unpaid consultant work at BMS, Genentech/Roche, Merck, and AstraZenecka. She receives clinical trial support from AstraZeneca/Medimmune, Bayer, BMS, Genentech/Roche, Helsinn, Merck, SeaGen, and Xcovery. Dr.Neal has received honoraria from CME Matters, Clinical Care Options CME, Research to Practice CME, Medscape CME, Biomedical Learning Institute CME, MLI Peerview CME, Prime Oncology CME, Projects in Knowledge CME, Rockpointe CME, MJH Life Sciences CME, Medical Educator Consortium, and HMP Education. He is a consultant at AstraZeneca, Genentech/Roche, Exelixis, Takeda Pharmaceuticals, Eli Lilly and Company, Amgen, Iovance Biotherapeutics, Blueprint Pharmaceuticals, Regeneron Pharmaceuticals, Natera, Sanofi/Regeneron, D2G Oncology, Surface Oncology, Turning Point Therapeutics, Mirati Therapeutics, Gilead Sciences, Abbvie, Summit Therapeutics, Novartis, Novocure, Janssen Oncology, and Anheart Therapeutics. He receives research funding from Genentech/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, Nektar Therapeutics, Takeda Pharmaceuticals, Adaptimmune, GSK, Janssen, AbbVie, and Novocure. Dr. Berry has received honoraria from Merck Pharmaceuticals for lectures on lung cancer biomarker testing for patients receiving pembrolizumab. Dr. Waliany is a consultant at AstraZeneca. * ICIs : immune checkpoint inhibitors MRL : Murphy Roths Large scRNAseq : single-cell RNA-sequencing scTCRseq : single-cell T-cell receptor sequencing CITE-seq : cellular indexing of transcriptomes and epitopes irAEs : immune related adverse events PD-1 : programmed cell death-1 CTLA-4 : cytotoxic T-lymphocyte antigen 4