Difference between revisions of "Part:BBa K4175011"

Revision as of 14:47, 9 October 2022

IL-6R-PD1

Sequence and Features

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.

Characterization

Figure 3. The schematic map of MND-IL-6R-PD-1-UAS-pSV40-aCD19CAR-P2A-mCherry plasmid.

Extracellular human IL-6R (aa 1-386, sequence found on Ensembl) and intracellular human PD-1 (aa 191-289, sequence found on Ensembl) was synthesized manually by GenScript. Then the synthesized IL-6R-PD-1 gene was cloned into MND plasmid with UAS-pSV40-aCD19CAR-P2A-mCherry (BBa_K4175012) using seamless cloning (Fig 3). Both the MND plasmid and anti-CD19 CAR was kindly provided by our primary PI, Huang He. Then we transfected Jurkat cells with the plasmid using nucleofection. As both MND and pSV40 are strong promoters, we expected that IL-6R-PD-1 and anti-CD19 CAR be constitutively expressed on the cell membrane. The expression of IL-6R-PD-1 and anti-CD19 CAR was confirmed using flow cytometry.

After obtaining IL-6R-PD-1 expressing CAR-T (NFL_Jurkat) cells, we incubated them with CD19+/luciferase+ Raji cells in a 3:1 ratio. To evaluate the capacity of IL-6R-PD-1 to inhibit CAR function under different concentrations of IL-6, we used culture media that contained 0 pg/ml, 1 pg/ml, 10 pg/ml, 100 pg/ml, and 1000 pg/ml IL-6, respectively, to incubate CAR-T and Raji. After co-incubation for 4h, 8h, 16h and 24h, each group of culture was added with the same dose of D-luciferin and the fluorescence intensity was measured to quantify the amount of surviving Raji cells.

Figure 3. The survival rate of Raji cells under different co-culture system (blue, Raji; orange, Raji cocultured with untransfected CAR-only Jurkat; gray, Raji cocultured with CAR/IL-6R-PD-1 dual-transfected Jurkat). The title of each panel represents IL-6 concentration (pg/ml).

It was found that under 100 pg/ml IL-6, IL-6R-PD-1 had a significant inhibitory effect on cytotoxicity through 2h to 24h after co-culture (Fig 4). Surprisingly, the inhibitory effect was not significant under 1000 pg/ml IL-6, as the survival rate of Raji under treatment with NFL_Jurkat and CAR-T without NFL (Jurkat) was fairly comparable until 24h. However, if we compared the NFL_Jurkat treatment group with the control group (Raji with no CAR-T cells), we found that the survival rate of Raji was similar in all the groups. This may suggest that IL-6R-PD-1 has a good inhibitory effect under low and high concentrations of IL-6. Moreover, the survival rate of Raji in NFL_Jurkat treatment group was much higher than Jurkat group after 24h co-culture whatever the IL-6 level was. Although we did seem to successfully inhibit the CAR using IL-6R-PD-1, the NFL_Jurkat failed to exert its cytotoxicity effect under low IL-6 level.

Discussion

In human, the normal serum level of IL-6 is ranged between 0 and 2.9 pg/ml. To assure that the anti-leukemic effect (in our project, it should be the senolytic effect) is not compromised, the PD-1 should not be activated within that range. Unfortunately, IL-6R-PD-1 seemed to still inhibit CAR function when IL-6 concentration was normal. The sensor for IL-6 should thus be replaced with a mutated version of IL-6R or anti-IL-6 scFv that has lower affinity for IL-6 than IL-6R. Also, the expression % of CAR in Jurkat after transfection in our experiment was not high enough. This may be responsible for lack of stable cytotoxicity of CAR-T cells against Raji cell lines in our experiments.

References

Barrett, D.M., Singh, N., Hofmann, T.J., Gershenson, Z., Grupp, S.A., 2016. Interleukin 6 Is Not Made By Chimeric Antigen Receptor T Cells and Does Not Impact Their Function. Blood 128, 654. https://doi.org/10.1182/blood.V128.22.654.654

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

Shimabukuro-Vornhagen, A., Gödel, P., Subklewe, M., Stemmler, H.J., Schlößer, H.A., Schlaak, M., Kochanek, M., Böll, B., von Bergwelt-Baildon, M.S., 2018. Cytokine release syndrome. J. Immunother. Cancer 6, 56. https://doi.org/10.1186/s40425-018-0343-9

Teachey, D.T., Lacey, S.F., Shaw, P.A., Melenhorst, J.J., Maude, S.L., Frey, N., Pequignot, E., Gonzalez, V.E., Chen, F., Finklestein, J., Barrett, D.M., Weiss, S.L., Fitzgerald, J.C., Berg, R.A., Aplenc, R., Callahan, C., Rheingold, S.R., Zheng, Z., Rose-John, S., White, J.C., Nazimuddin, F., Wertheim, G., Levine, B.L., June, C.H., Porter, D.L., Grupp, S.A., 2016. Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia. Cancer Discov. 6, 664–679. https://doi.org/10.1158/2159-8290.CD-16-0040