Blocking MIF secretion enhances CAR T-cell efficacy against neuroblastoma

While chimeric antigen receptor (CAR) T-cell therapies are showing highly promising first results in neuroblastoma, immunosuppressive tumor microenvironments (TME) limit T cell persistence and durable clinical efficacy. To improve CAR T-cell efficacy further, we applied a multi-omics approach including single-cell RNA sequencing and proteomics, which identified 13 targetable immunosuppressive factors in neuroblastoma. Of these, macrophage migration inhibitory factor (MIF) and midkine (MDK) were validated across multiple published RNA datasets. Moreover, they were secreted in high abundance by neuroblastoma tumoroids. Functional validation experiments revealed MIF as a potent inhibitor of CAR T-cells, in vitro and in vivo. Degradation of MIF by PROTAC technology significantly enhanced CAR T-cell activation targeting GPC2 and B7-H3, providing a potential intervention against MIF. By defining the immunosuppressive effects of neuroblastoma's TME on CAR T-cell efficacy, particularly the pivotal role of MIF, we provide a therapeutic strategy for improving adoptive cell therapies for this pediatric malignancy. ### Competing Interest Statement K.R.B. and G.P.P have applied for patents for the discovery and development of immunotherapies for cancer, including patents related to GPC2-directed immunotherapies. K.R.B receives royalties from Tmunity/Kite Pharma and ConjugateBio, Inc. for licensing of GPC2-related technology and funding from Tmunity/Kite Pharma and ConjugateBio, Inc. for research on GPC2-directed immunotherapies. K.R.B. is on the ConjugateBio Scientific Advisory Board. M.B. holds patents pertinent to cellular immunotherapy development and manufacture, and has consulted for Lava Therapeutics. J.A. holds founder stock in Autolus ltd, consults for Roche and BMS, and holds patents in CAR-T design. J.M. has received research funding from Roche for in vitro work. No other conflicts of interest are declared.