Part:BBa_K4175003

human intracellular PD-1

This part is the intracellular domain of human programmed death-1 (PD-1) (aa 191-289).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

PD-1 acts as an inhibitory ‘checkpoint’ upon T cell activation to prevent T cells from attacking healthy cells (Sharpe and Pauken, 2018). In the early phase of T cell activation, PD-1 is expressed on the membrane of effector T cells. When programmed death-ligand 1 (PD-L1) and PD-L2 on the antigen-presenting cells (APCs) bind to PD-1, the immunoreceptor tyrosine-based switch motif (ITSM) in the intracellular part of PD-1 will recruit phosphatases such as SHP2 (Fig 1). These phosphatases can then disturb the positive signal exerted by T cell receptors (TCR) by inhibiting ZAP70, and PI3K-AKT and RAS signaling pathway. Notably, the TCR signaling can only be inhibited by PD-1 when PD-L1 or PD-L2 is presented on the same cell where the antigen for TCR recognition is presented (Sharpe and Pauken, 2018). As a result, the activation of various transcriptional factors, such as AP-1, NFAT and NF-κB is decreased, leading to decreased T cell activation, proliferation and survival. It has also been found that PD-1 can trigger basic leucine zipper transcriptional factor ATF-like (BATF) activation, which has negative effect on TCR signaling (Sharpe and Pauken, 2018).

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

Usage

In a published report, Fedorov et al. joined intracellular part of PD-1 (aa 145 to 288) with PSMA scFv, CD8 hinge and CD8 transmembrane domain to create an inhibitory CAR (iCAR). They also use CTLA-4, which is another co-inhibitory receptor on T cells, to create iCAR. Either of these iCARs was then co-expressed with anti-CD19 CAR (19-28z) on T cells. These T cells were subsequently co-incubated with CD19+/PSMA- fibroblasts (target) or CD19+/PSMA+ fibroblasts (off-target). After 120 hours co-incubation, it was found that PD-1-iCAR has a strong inhibitory effect on T cell cytotoxicity when PSMA+ cells are present (Fedorov et al., 2013) (Fig 2).

Figure 2. (Fedorov et al., 2013) Different groups of T cells were incubated with target (CD19+/PSMA-) or off-target (CD19+/PSMA+) cells in a 1:1 ratio. mCherry is expressed in these target and off-target cells. Pdel represents T cells with mock iCAR (only has anti-PSMA scFv but lacks PD-1 or CTLA4). N/A represents T cells with only anti-CD19 CAR.

19-28z/PD-1 iCAR T cells were also incubated with 1:1 CD19+/GFP+ (target) and CD19+/PSMA+/mCherry+ (off-target) fibroblasts. After 38h incubation, the target cells were almost completely eliminated while the number of off-target cells increased. In comparison, 19-28z CAR-T cells with mock iCAR(19-28z/Pdel) killed the target and off-target cells at almost the same proportion (Fig 3). This showed that PD-1 iCAR T cells were able to distinguish between target and off-target cells (Fedorov et al., 2013). In vivo experiment had also proved that PD-1 iCAR can inhibit T cell cytotoxicity specifically when off-target cells are present (Fig 4).

Figure 3. (Fedorov et al., 2013) T cells were coincubated with 1:1 target (GFP-expressing) and off-target cells (mCherry-expressing) for 38 h. 19-28z/PD-1-iCAR was shown to selectively kill target but not off-target cells.

Figure 4. (Fedorov et al., 2013) 1:1 NALM/6 (target) and NALM/6-PSMA (off-target) cells were injected into NOD/SCID/γc- mice. Then these mice were treated with either 19-28z (without iCAR) or 19-28z/PD-1 iCAR T cells. Using flow cytometry, the proportion of target and off-target cell was analyzed. The left gate represents target cells, while the right gate represents the off-target cells.

For our usage, we joined intracellular part of PD-1 (aa 191-289) with extracellular and transmembrane part of IL-6R (aa 1-386) (BBa_K4175002). We expected that this synthetic device will work in such a way that when serum IL-6 level is high, PD-1 will be triggered to inhibit the CAR signaling. In this way, the IL-6 level will stop further increase and prevent the development of severe cytokine release syndrome (CRS). For more detailed information, please see BBa_K4175011.

Figure 5. IL-6R-PD-1 in CAR-T cells under low and high IL-6 concentration.

Reference

Fedorov, V.D., Themeli, M., Sadelain, M., 2013. PD-1– and CTLA-4–Based Inhibitory Chimeric Antigen Receptors (iCARs) Divert Off-Target Immunotherapy Responses. Sci. Transl. Med. 5, 215ra172. https://doi.org/10.1126/scitranslmed.3006597

Sharpe, A.H., Pauken, K.E., 2018. The diverse functions of the PD1 inhibitory pathway. Nat. Rev. Immunol. 18, 153–167. https://doi.org/10.1038/nri.2017.108