Chimeric antigen receptor (CAR) T cell therapy is hindered in solid tumor treatment due to the immunosuppressive tumor microenvironment and suboptimal T cell persistence. Current strategies do not address nutrient competition in the microenvironment. Hence, we present a metabolic refueling approach using inosine as an alternative fuel. CAR T cells were engineered to express membrane-bound CD26 and cytoplasmic adenosine deaminase 1 (ADA1), converting adenosine to inosine. Autocrine secretion of ADA1 upon CD3/CD26 stimulation activates CAR T cells, improving migration and resistance to transforming growth factor β1 suppression. Fusion of ADA1 with anti-CD3 scFv further boosts inosine production and minimizes tumor cell feeding. In mouse models of hepatocellular carcinoma and non-small cell lung cancer, metabolically refueled CAR T cells exhibit superior tumor reduction compared to unmodified CAR T cells. Overall, our study highlights the potential of selective inosine refueling to enhance CAR T therapy efficacy against solid tumors.

Chimeric antigen receptor (CAR) T cell therapy represents a cutting-edge cancer treatment, making the development and testing of CAR T cells crucial for advancing this therapeutic strategy. We present a protocol for creating and characterizing human epidermal growth factor receptor 2 (HER2)- and glypican-3 (GPC3)-metabolic reprogramming (MR)-CAR T cells by overexpressing adenosine deaminase 1 (ADA1) and CD26 (also known as dipeptidylpeptidase-4 or DPP4). This approach effectively converts immunosuppressive adenosine into inosine, which supports T cell survival in glucose-deficient tumor microenvironments. The protocol includes producing retroviral vectors, generating CAR T cells, and conducting ecto-ADA1 activity, cytotoxicity, cell migration, and RNA sequencing assays. For complete details on the use and execution of this protocol, please refer to Hu et al.¹.

Background/Objectives: Chimeric antigen receptor (CAR) T cells are a powerful cancer therapy, but their function depends heavily on internal signaling domains and metabolic adaptability. Most studies evaluate CAR behavior upon antigen exposure, yet intrinsic signaling properties may pre-program CAR T cell states even in the absence of stimulation. This study investigates how CAR design and metabolic support shape baseline transcriptional programs, focusing on tonic signaling and NF-κB-related pathways. Methods: We engineered CAR T cells targeting HER2 or GPC3 antigens, incorporating either 4-1BB or CD28 co-stimulatory domains, respectively. A subset of cells was further modified with adenosine deaminase 1 (ADA1) and CD26 to degrade extracellular adenosine and supply inosine, a metabolic strategy termed metabolic refueling (MR). Bulk RNA-seq was performed on resting T cells without antigen stimulation. We analyzed differential gene expression, gene set enrichment (GO, KEGG, Hallmarks), and transcription factor activity (DoRothEA) to assess the impact of CAR design and MR on T cell programming. Results: All CAR T cells exhibited activation of NF-κB–centered inflammatory programs at baseline, indicating tonic signaling. GPC3 CAR T cells showed stronger baseline activation than HER2 CAR T cells. Metabolic refueling amplified these programs without altering their directionality, enhancing inflammatory, survival, and effector modules. Transcription factor activity scores mirrored these trends, highlighting RELA, FOS, and STATs as key regulatory nodes. Conclusions: CAR-intrinsic features, notably co-stimulatory domain choice, define the tonic NF-κB activation tone in resting CAR T cells. Metabolic refueling boosts these baseline states without overstimulation, suggesting it may be especially valuable for weaker CAR constructs. These findings provide a framework for tuning CAR T cell function through combinatorial design strategies targeting signaling and metabolism.

Background: Chimeric antigen receptor (CAR) T-cell therapy for solid tumors is limited by inadequate T-cell persistence and tumor infiltration, as well as treatment-associated cytokine release syndrome (CRS). To address these challenges, we developed a metabolically rewired CAR T-cell (MRCAR) platform by co-expressing adenosine deaminase 1 (ADA1) and CD26 in CAR T cells. Methods: MRCAR T cells were generated using either GPC3-targeted CARs for hepatocellular carcinoma (HCC) or HER2-targeted CARs for breast cancer. For HCC, we further evaluated constructs with and without enforced IL-15 expression to boost T-cell function. Results: In vitro, IL-15–expressing GPC3-MRCAR T cells exhibited enhanced proliferation, sustained effector function, reduced exhaustion, and superior cytotoxicity following repeated antigen stimulation compared with conventional CAR T cells. CD26 expression significantly enhanced CAR T-cell invasiveness in transwell assays and promoted tumor infiltration in vivo in both tumor models. ADA1 expression enhanced CAR T-cell persistence and was associated with increased frequencies of memory and stem-like T-cell populations in vivo. Notably, although IL15-GPC3-CAR T-cell treatment increased antitumor activity, it also caused high mortality and systemic inflammation in Huh7 tumor-bearing mice, whereas incorporation of ADA1 and CD26 into MRCAR T cells significantly improved survival and reduced serum levels of IL-6, IFN-γ, and TNF-α. Mechanistically, conventional IL-15–expressing GPC3-CAR T cells induced proinflammatory M1-like macrophage activation, whereas MRCAR T cells markedly suppressed this inflammatory response. Conclusions: Collectively, these results indicate that metabolic rewiring via ADA1 and CD26 enhances CAR T-cell expansion, tumor infiltration, persistence, and safety, supporting the MRCAR platform as a promising strategy to improve both efficacy and tolerability of CAR T-cell therapy for solid tumors.

Chimeric antigen receptor (CAR) T-cell therapy for solid tumors is limited by inadequate T-cell persistence and tumor infiltration, as well as treatment-associated cytokine release syndrome (CRS). To address these challenges, we developed a metabolically rewired CAR T-cell (MRCAR) platform by co-expressing adenosine deaminase 1 (ADA1) and CD26 in CAR T cells. MRCAR T cells were generated using either GPC3-targeted CARs for hepatocellular carcinoma (HCC) or HER2-targeted CARs for breast cancer. For HCC, we further evaluated constructs with and without enforced IL-15 expression to boost T-cell function. In vitro, IL-15–expressing GPC3-MRCAR T cells exhibited enhanced proliferation, sustained effector function, reduced exhaustion, and superior cytotoxicity following repeated antigen stimulation compared with conventional CAR T cells. CD26 expression significantly enhanced CAR T-cell invasiveness in transwell assays and promoted tumor infiltration in vivo in both tumor models. ADA1 expression enhanced CAR T-cell persistence and was associated with increased frequencies of memory and stem-like T-cell populations in vivo. Notably, although IL15-GPC3-CAR T-cell treatment increased antitumor activity, it also caused high mortality and systemic inflammation in in Huh7 tumor-bearing mice, whereas incorporation of ADA1 and CD26 into MRCAR T cells significantly improved survival and reduced serum levels of IL-6, IFN-γ, and TNF-α. Mechanistically, conventional IL-15–expressing GPC3-CAR T cells induced proinflammatory M1-like macrophage activation, whereas MRCAR T cells markedly suppressed this inflammatory response. Collectively, these results indicate that metabolic rewiring via ADA1 and CD26 enhances CAR T-cell expansion, tumor infiltration, persistence, and safety, supporting the MRCAR platform as a promising strategy to improve both efficacy and tolerability of CAR T-cell therapy for solid tumors.

Background: Retroviral vectors remain widely used for CAR T-cell manufacturing, including emerging multi-component engineering strategies that incorporate additional functional modules to enhance therapeutic performance. However, genome-wide profiling of integration patterns in CAR T cells subjected to multiplex retroviral engineering has not previously been reported. Methods: We performed genome-wide profiling of retroviral integration sites in CAR T-cell products derived from two targeting platforms (IL15-GPC3 and HER2). Each platform was evaluated under conditions with or without co-transduction of a retroviral vector encoding ADA1 and CD26. Experiments were conducted using cells obtained from a single donor with three technical replicates. Vector copy number (VCN), chromosomal distribution, genomic feature annotation, and integration hotspot analyses were conducted. Results: VCN levels were comparable across all CAR T-cell products and were not significantly affected by ADA1/CD26 co-expression. Integration events were broadly distributed across the genome with enrichment in gene-dense chromosomal regions. Chromosome 19 showed a prominent integration preference, with a secondary enrichment observed on chromosome 17. The majority of integration sites were located within intronic regions, with no enrichment observed in promoter regions. Recurrent integration hotspots were identified; however, no evidence of dominant clonal expansion was observed across conditions. Comparative analyses between ADA1/CD26 and control groups showed minimal differences in integration patterns. Conclusions: To our knowledge, this study provides the first comprehensive, genome-wide characterization of retroviral integration site profiles in CAR T-cell products manufactured by multi-vector co-transduction with an ADA1/CD26 module. By employing harmonized comparison with external reference datasets, we offer a directly benchmarked integration site characterization of multi-component CAR T-cell manufacturing. These findings establish a reference framework for integration site monitoring in next-generation CAR T-cell engineering systems and inform risk evaluation for emerging combinatorial vector strategies.