Blocking MIF secretion enhances CAR T-cell efficacy against neuroblastoma.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy is a promising and innovative cancer therapy. However, immunosuppressive tumor microenvironments (TME) limit T cell persistence and durable efficacy. Here, we aimed to identify and target immunosuppressive factors in the TME of neuroblastoma, a pediatric extracranial solid tumor, to improve CAR-T efficacy. METHODS: Immunosuppressive factors were identified using a multi-omics approach, including single-cell RNA sequencing (scRNA-seq) of 24 neuroblastoma tumors, published bulk-RNA sequencing datasets, and mass-spectrometry of patient-derived tumoroid models. Candidate targets were validated with functional assays in vitro and in vivo. Protein degradation of the top immunosuppressive target by PROTAC technology was used to evaluate the effect on CAR T-cell activity. RESULTS: ScRNA-seq revealed 13 immunosuppressive interactions in the TME of neuroblastoma, two effectors of which, Midkine (MDK) and Macrophage Migration Inhibitory Factor (MIF), were validated as candidate targets across multiple published datasets. Both factors were among the top 6 % of most abundantly secreted factors by patient-derived tumoroid models, substantiating their potential relevance in the TME. In vitro and in vivo functional assays confirmed MIF to be a potent inhibitor of CAR T-cell activation and killing capacity. To translate these findings into a potentially clinically applicable treatment, we explored MIF targeting by PROTAC technology, which significantly enhanced activation of CAR T-cells targeting GPC2 and B7-H3. CONCLUSION: By defining the immunosuppressive effects of neuroblastoma's TME on CAR T-cell efficacy, revealing the pivotal role of MIF, we provide an analytic pipeline and therapeutic strategy for improving adoptive cell therapies for this pediatric malignancy and potentially other solid tumors.