Rapid mRNA engineering of cytokine-armored CAR immune cell communities exhibits promising preclinical therapeutic activity against glioblastoma

Chimeric antigen receptor (CAR) cell therapies for solid tumors remain constrained by costly and time-intensive manufacturing of single-lineage products, and limited activity within immunosuppressive tumor microenvironments. Here, we developed a rapid, cost-efficient, non- viral manufacturing platform that generates multifunctional and multi-lineage CAR immune cell communities by mRNA electroporation into non-activated, non-expanded peripheral blood mononuclear cells (PBMC) and applied it to glioblastoma. We engineered the cells with unconventional multi-targeting NKG2D-based or classical scFv-based CAR constructs, incorporated different cytokine payloads and evaluated their anti-tumor activity, contributions of different immune cell types, transcriptional states and effects on the microenvironment across in vitro assays, orthotopic glioma mouse models and human glioma-bearing organotypic brain slices. The rapid production process yielded over 90% viability and efficient CAR expression across T cell, NK cell, and monocyte compartments without altering baseline immune cell composition. Cytokine armoring markedly enhanced tumor cell killing in vitro and induced payload-specific immune effector transcriptional programs. In orthotopic glioma mouse models, multifunctional CAR-PBMC reduced tumor burden and prolonged survival with partly durable responses. In human organotypic glioma-bearing brain slices, the rapidly produced CAR immune cell communities infiltrated the tumor-bearing tissue, limited glioma outgrowth and increased tumor cell apoptosis with limited effects on non-malignant cells such as neurons, astrocytes and microglia. Armoring the cells with IL-2/IL-15 (Cyto1) favored cytotoxic and memory-associated T cell and NK cell programs, whereas IL-12/IFNα2 (Cyto2) elicited a broader interferon-dominant inflammatory program engaging stromal and myeloid compartments. Together, these findings establish mRNA engineering of minimally manipulated PBMC as a rapid modular strategy with potential for point-of-care adaptation for glioblastoma and expandable to other solid tumors.