{
  "abstract": "Background Barriers to successful CAR-T cell therapy include exhaustion and lack of persistence, competitive metabolic demands, and associated CAR-T cell-driven toxicities. Responses are often characterized by rapid expansion accompanied by a robust and multi-dimensional pro-inflammatory state. CAR-T cells currently lack a means of control. In contrast, T-cell receptor (TCR) engagement results in productive activation yet is balanced by immune checkpoint receptors which regulate activity and maintain immune homeostasis. Our goal is to temper the CAR-T cell response by introducing a system of autoregulation to counter the potent activation delivered via the CAR receptor. We designed a chimeric inhibitory receptor (CIR), consisting of an IFN-gamma binding domain coupled to the inhibitory signaling domain of TIGIT, to provide negative signaling in response to high levels of CAR activation and IFN-g ( figure 1A).Methods An anti-IFN-g scFv followed by the intracellular domain of TIGIT was co-expressed with a CD19 CAR and either a CD28 or 4-1BB costimulatory domain. Gene expression profiling was performed using Nanostring nCounter analysis. Activation state following stimulation with CD19 + NALM-6 tumor cells was evaluated by flow cytometry. Single-cell secretome measurements were evaluated by Isolight analysis. 6-8-week-old NSG mice were injected with NALM-6 tumor cells and two days later with CAR-T cells.Results Gene expression analysis of CAR.CIR T cells revealed attenuation across multiple major T-cell signaling pathways including JAK-STAT, PI3-Kinase, and NFAT with an accompanying shift in metabolic re-programming relative to CD19 controls following stimulation by tumor cells ( figure 1B). Decreased activation was confirmed by a reduction in cells expressing multiple markers of activation (figure 2A). Despite attenuation, CIR-expressing CD19 CARs exhibited normal activity in cytotoxicity assays. Monocyte-derived macrophage and dendritic cell activation by CAR.CIR T cells were reduced as evidenced by decreased secretion of myeloid cell-derived proinflammatory mediators. Preliminary secretome analysis suggests improved polyfunctionality of CAR.CIR T cells, an indicator of increased clinical efficacy. CAR.CIR T cells also exhibited a reduction in PD-1, LAG-3, and TIM-3 following in vitro continuous antigen exposure (figure 2B), suggesting improved resistance to exhaustion. Importantly, in vivo anti-tumor responses were not adversely affected as CAR.CIR T cells efficiently controlled disease in leukemia-bearing mice (figure 2C). Similar results were recapitulated in 4-1BB-costimulated CARs, highlighting broader potential clinical applicability.Conclusions Self-regulation in CAR-T cells may confer advantages such as tempered activation, improved functionality and metabolic fitness, and enhanced persistence. These results provide a compelling proof of concept of an innovative auto-regulating CAR-T cell design with the potential to improve responses and mitigate severe toxicities.Ethics Approval Human PBMC collection from healthy donors was performed according to an Institutional Review Board approved protocol. UVA-HSR-IRB #18842 All animal studies were conducted under a protocol approved by and in compliance with policies set by the Institutional Animal Care and Use Committee (IACUC). Protocol #4189Abstract 213 Figure 1A) Schematic diagram and proposed model of an auto-regulating CD19 CAR-T cell expressing an anti-IFN-gamma scFv coupled to the TIGIT inhibitory receptor signaling domain. B) Nanostring nCounter gene expression profiling of CD19.28z.CIR CAR-T cells following stimulation by NALM-6 tumor cells reveals substantial attenuation across multiple activation pathways and major shifts in metabolic activity. Nanostring nCounter gene expression profiling of isolated RNA was performed following tumor cell challenge using the CAR-T Characterization Panel. Standard quality control and data normalization was completed in the ROSALIND platform followed by individual pathway analysis and differential expressionAbstract 213 Figure 2A) CD19 CAR or CD19 CAR.CIR T cells were cultured with or without NALM-6 for 24 hours then activation was evaluated by flow cytometry. B) CD19 CAR and CD19 CAR/CIR T cells were subjected to repeated stimulation by NALM-6 cells every 2-3 days at a 1:4 E:T ratio. Exhaustion marker analysis was performed on day 12 by flow cytometry. C) On day -2, NSG mice were injected with 0.5x106 NALM-6. 2 days later, mice were treated i.v. with CD19 or CD19.CIR CAR-T cells (10x106). BLI for disease burden was performed 2-3 times/week. Representative of 2 individual experiments; n=4 mice/group",
  "authors": [
    {
      "affiliations": [
        "University of Virginia, Crozet, VA, USA"
      ],
      "name": "Dustin A Cobb"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Lixia Liu"
    },
    {
      "affiliations": [
        "University of Virginia School of Medicine, Charlottesville, VA, USA"
      ],
      "name": "Amanda M Lulu"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Daniel Lee"
    }
  ],
  "title": "213 Development of ‘auto-regulating’ CD19 CAR T cells using a novel chimeric inhibitory receptor",
  "uid": "e1a1a3e8-f330-5c4e-93b1-94293fcc8410"
}