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Background Antigen escape is one of the leading causes of relapse following chimeric antigen receptor (CAR)-T therapy, particularly in multiple myeloma. A critical gap persists in understanding the tumor-intrinsic pathways that trigger antigen loss, insight essential for devising strategies to resensitize tumors to immune attack. We identify a previously uncharacterized post-translational mechanism centered on the metabolic enzyme ribonucleotide reductase subunit M2 (RRM2), termed trafficking-mediated antigen escape, to enhance cellular therapy efficacy.Methods We combined single-cell RNA sequencing analysis with multiplex immunofluorescence to identify a clinically relevant RRM2 + myeloma subpopulation exhibiting low MICA/B abundance. Functional validation included induced pluripotent stem cell-derived myeloma organoids monitored by real-time imaging and disseminated xenograft models to assess the effect of subtoxic osalmid treatment on NKG2D CAR-T cell activity. Co-immunoprecipitation, guanosine 5′-triphosphate pulldown, and confocal microscopy were used to investigate the underlying trafficking mechanism.Results Single-cell analysis uncovered a clinically prevalent RRM2 + myeloma subpopulation with profoundly reduced MICA/B surface abundance, which established tumor-intrinsic heterogeneity as one of fundamental causes of NKG2D CAR-T resistance. We further demonstrated RRM2’s non-canonical role as a trafficking regulator that actively shuttles MICA/B toward lysosomal degradation via RAB7A activation while simultaneously blocking RAB11-mediated recycling. Therapeutic intervention using subtoxic osalmid, a clinically approved drug and previously characterized as an RRM2 inhibitor, successfully reversed this trafficking defect, restored MICA/B membrane presentation and synergized with NKG2D CAR-T cells to enhance their expansion, polyfunctional cytokine secretion, and stem-like properties. This combination strategy achieved durable tumor remission in vivo by sustaining T-cell fitness while reducing exhaustion, offering an immediately actionable solution to clinical antigen escape.Conclusions Our study establishes RRM2-driven trafficking as a novel and targetable mechanism of antigen escape in CAR-T therapy. By repurposing osalmid to restore MICA/B surface presentation, we provide a clinically translatable strategy that specifically potentiates NKG2D CAR-T cell efficacy in multiple myeloma and could potentially enhance the efficacy of CAR-T across diverse antigens. This work highlights the therapeutic potential of modulating intracellular trafficking to overcome resistance in cellular immunotherapy.