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Background The aim of this study was to validate the manufacturing of CD19-CAR-T cells generated from umbilical cord blood (UCB) and perform the comprehensive multi-omics and functional characterization.Methods T cells were isolated from umbilical cord blood (UBCs; N=20) and activated in vitro using CD3 and CD28 monoclonal antibodies (mAbs). These cells were then transduced with lentiviral vectors encoding CD19-CD28z or CD19-4-1BBz chimeric antigen receptors (CARs). As a reference, engineered T cells were also produced from peripheral blood lymphocytes (PBL; N=8). The CD19-CAR-T cells were co-incubated with either CD19+, to induce antigen-mediated CAR engagement, or CD19- target cells. Subsequently, multi-omics analyses, including metabolomics, transcriptomics, single-cell RNA sequencing (scRNAseq), methylation profiling, and in vitro functional assays (Elispot and Luminex), were performed.Results The multi-omics characterization, paired with functional assays, identified distinct features based on the source of T cells used for manufacturing CD19-CAR-T cells. Transcriptomic profiling revealed that pathways related to chemotaxis, adhesion, and activation were up-regulated in UCB-CD19-CAR-T cells compared to PBL-CD19-CAR-T cells. Molecules involved in inflammation, allograft rejection, graft vs. host disease (GvHD), and antigen presentation were overexpressed in PBL-CD19-CAR-T cells compared to UCB-CD19-CAR-T cells. A TH1-type profile was up-regulated in UCB-CD19-CAR-T cells, while pro-inflammatory molecules (e.g., IL-9, IL-10, IL-13, IL-31) were preferentially associated with PBL-CD19-CAR-T cells. These findings were confirmed at protein-level analyses upon the antigen-specific engagement of the CD19-CAR. Differential metabolomic profiles were also identified following antigen-specific vs. antigen-independent stimulation of CD19-CAR-T cells, including amino acid, amino sugar metabolism, amino protein biosynthesis and aminoacyl-tRNA biosynthesis pathways, with the latest being involved in T cell activation and maturation, and in preventing cytokine release syndrome. The scRNA sequencing identified transcripts specifically associated with the reactivity of either CD4 + or CD8+ CD19-CAR-T cells upon the co-culture with CD19+ target cells (lymphoma cell line) and highlighted the cell subtype complexity in association with anti-tumor functions. Along this line, the integration of the obtained results with the methylation profiles, allowed to obtain a deep genomic characterization and identify molecular pathways that are responsible of persistence and maturation of T cells.Conclusions The comprehensive multi-omics characterization of UCB-CD19-CAR-T cells highlighted their potent anti-tumor efficacy, reduced pro-inflammatory profile, and potentially enhanced safety. These findings support the development of UCB-derived ‘off-the-shelf’ CAR-T cells to make these innovative therapies more accessible to a broader number of cancer patients and more sustainable for the healthcare system.Ethics Approval This study was approved by the Sidra Medicine’s Ethics Board, #1812044429