{
  "abstract": "Background Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells is a revolutionary treatment in cancer therapy. CAR T therapy has achieved remarkable success in hematological B cell malignancies. However, it has faced significant challenges in solid tumors due to various factors, such as complex tumor microenvironments, restricted trafficking, impersistent antitumor activity and toxicities. A better understanding of CAR T biology will accelerate development of CAR T therapies with improved antitumor efficacy, durability and decreased toxicities.High-parameter cytometry is a powerful tool to functionally characterize CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. Fluorescence-based cytometry faces significant challenges with signal overlap and autofluorescence, limiting sensitivity and the number of targets detected in CAR T cells. Consequently, rare cell populations and functional readouts of CAR T cells are difficult to resolve. CyTOF™ technology overcomes these limitations with low signal spillover and absence of autofluorescence. To minimize technical variation, metal-tagged antibody cocktails and stained cell samples can be frozen for later use and acquisition, enabling a streamlined and flexible workflow in clinical research. Here, we present a 40-plus-marker CyTOF panel to simultaneously analyze phenotypic and functional protein expression in CAR T cells from in vitro co-culture with tumor cells.Methods CD19-targeted CAR T cells were expanded in vitro and co-cultured with Nalm6 cells at an E:T (effector cell: target cell) ratio of 1:3 for 2–4 days. A high-parameter CyTOF panel including 43 surface, cytoplasmic and nuclear markers was used to stain CAR T cells. The co-culture samples collected at different time points were stained using a pre-aliquoted frozen antibody cocktail following surface and intracellular (simultaneous cytoplasmic and nuclear targets) staining procedures. Stained samples were frozen and simultaneously acquired on a CyTOF XT system later.Results The cytotoxicity, activation, proliferation, differentiation and exhaustion of CAR T cells were evaluated. Comprehensive profiling revealed that CAR T cells became activated, proliferated and produced cytokines in in vitro co-culture with tumor cells and exhibited an exhaustive-like Treg phenotype at the end of a four-day co-culture. A diverse polyfunctional antitumor signature in the CD8 TEMRA cell subset was discovered during the co-culture.Conclusions Overall, we demonstrate that the high-parameter CyTOF panel enables deep functional characterization of CAR T cells by simultaneous detection of surface, cytoplasmic and nuclear markers, supporting advancing clinical development of CAR T therapies.For Research Use Only. Not for use in diagnostic procedures.",
  "authors": [
    {
      "affiliations": [
        "Standard BioTools, Markham, ON, Canada"
      ],
      "name": "Ling Wang"
    },
    {
      "affiliations": [
        "Standard BioTools, Markham, ON, Canada"
      ],
      "name": "David Howell"
    },
    {
      "affiliations": [
        "Standard BioTools, Markham, ON, Canada"
      ],
      "name": "Deeqa Mahamed"
    },
    {
      "affiliations": [
        "Standard BioTools, Markham, ON, Canada"
      ],
      "name": "Lauren J Tracey"
    },
    {
      "affiliations": [
        "Standard BioTools, South San Francisco, CA, USA"
      ],
      "name": "Christina Loh"
    }
  ],
  "title": "283 Functional profiling of CAR T cells using high-dimensional CyTOF: integrating cytokine, transcription factor and immune checkpoint marker signatures",
  "uid": "58b9d10a-1fbd-5987-8c61-8e909aea765f"
}