{
  "abstract": "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",
  "authors": [
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Salim Bougarn"
    },
    {
      "affiliations": [
        "Jackson Laboratories, Farmington, CT, USA"
      ],
      "name": "Mohammed Toufiq"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Mohammed El-Anbari"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Shana Jacob"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Saroja Kotegar Balayya"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Evonne C Chin-Smith"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Issam Tout"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Fazulur Rehaman"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Lisa Mathew"
    },
    {
      "affiliations": [
        "NIH-NCI, Doha, Qatar"
      ],
      "name": "Kun Wang"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Li Liu"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Oleksandr Soloviov"
    },
    {
      "affiliations": [
        "Sidra Medical and Research Center, Doha, Qatar"
      ],
      "name": "Sara Tomei"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Damilola Olagunju"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Deepa Subramanian"
    },
    {
      "affiliations": [
        "Sidra Medicine, Doha, Qatar"
      ],
      "name": "Suruchi Mohan"
    },
    {
      "affiliations": [
        "IRCCS San Raffaele Scientific Institute, Milan, Italy"
      ],
      "name": "Chiara Bonini"
    },
    {
      "affiliations": [
        "IRCCS San Raffaele Scientific Institute, Milan, Italy"
      ],
      "name": "Monica Casucci"
    },
    {
      "affiliations": [
        "Jackson Laboratories, Farmington, CT, USA"
      ],
      "name": "Damien Chaussabel"
    },
    {
      "affiliations": [
        "IRCCS Ospedale Policlinico San Martino, Genova, Italy, Italy"
      ],
      "name": "Antonio Uccelli"
    },
    {
      "affiliations": [
        "Sidra Medicine, Genova, Italy"
      ],
      "name": "Cristina Maccalli"
    }
  ],
  "title": "250 Umbilical cord blood-derived CD19-CAR-T cells exhibit effective anti-tumor activity and demonstrate a low profile of pro-inflammatory and allograft rejection",
  "uid": "7555a4c8-3bc3-50eb-8f3c-a0a919a522c2"
}