Part:BBa_K4175011

IL-6R-PD1

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 60
  Illegal XbaI site found at 271
  Illegal PstI site found at 55
  Illegal PstI site found at 1117
  Illegal PstI site found at 1151
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 60
  Illegal PstI site found at 55
  Illegal PstI site found at 1117
  Illegal PstI site found at 1151
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 60
  Illegal BamHI site found at 76
  Illegal XhoI site found at 19
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 60
  Illegal XbaI site found at 271
  Illegal PstI site found at 55
  Illegal PstI site found at 1117
  Illegal PstI site found at 1151
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 60
  Illegal XbaI site found at 271
  Illegal PstI site found at 55
  Illegal PstI site found at 1117
  Illegal PstI site found at 1151
  Illegal NgoMIV site found at 669
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Figure 1.IL-6R-PD-1 in CAR-T cells under low and high IL-6 concentration.

Figure 2. (Sharpe and Pauken, 2018) The mechanism of PD-1 inhibition of T cell activity.

This device consists of the extracellular part of human IL-6R (aa 1-386) (BBa_K4175002) and the intracellular domain of human PD-1 (aa 191-289) (BBa_K4175003) (Fig 1).

PD-1 is an inhibitory receptor on the effect T cells that regulate TCR signaling upon T cell activation (Sharpe and Pauken, 2018). When PD-1 binds to its ligand PD-L1 or PD-L2 on the antigen-presenting cells, the intracellular domain of PD-1, ITSM, would recruit phosphatases like SHP2 (Fig 2). Then SHP2 inhibits ZAP70, and PI3K-AKT and RAS pathways to disturb the TCR signaling. This would downregulate various transcriptional factors (i.e., AP-1, NFAT, NF-κB) so that T cell activation, growth, proliferation, and survival is inhibited. In these ways, PD-1 acts as an inhibitory ‘checkpoint’ for T cell cytotoxicity against target cells instead of healthy cells (Sharpe and Pauken, 2018).

During CAR-T therapy, the most common side effect is cytokine release syndrome (CRS), which is characterized by a sharp increase in serum IL-6 level (Shimabukuro-Vornhagen et al., 2018; Teachey et al., 2016). IL-6 contributes to many symptoms of CRS such as vascular leakage and complement activation, but is not critical for the cytotoxic effect of T cells (Barrett et al., 2016; Shimabukuro-Vornhagen et al., 2018).

To prevent severe CRS during CAR-T treatment, we aimed to maintain IL-6 in a range that will not cause immune overaction. Because IL-6 is majorly secreted by monocytes/macrophages cell line in response to damage-associated molecular patterns (DAMPs) released by pyroptotic target cells after CAR-T killing, we intended to take advantage of the inhibitory properties of PD-1 to create a switch in CAR-T that would ‘turn off’ the ‘killing mode' when there is too much IL-6 in the serum. We joined the extracellular part of human IL-6R (sensor) with the intracellular part of human PD-1 (switch). We expected that under high concentration of serum IL-6, binding of IL-6 to IL-6R will trigger PD-1 activation (Fig 1). Through SHP2 recruitment, the CAR signaling will be terminated so that CAR-T will pause its cytotoxicity effect. Subsequently, less pyroptosis of target cells will be induced by CAR-T so that less IL-6 will be produced and serum IL-6 level will gradually decrease. Conversely, when the serum IL-6 level is low, IL-6 will be dissociated from the IL-6R due to the low affinity of receptor. PD-1 will thus stop inhibiting CAR signaling so that the CAR-T cell will continue its cytotoxic function (Fig 1). We hoped that this negative feedback loop (NFL) will maintain the serum level of IL-6 into a normal range.