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IL-6_scfv-Notch-Gal4KRAB

Sequence and Features

Usage and Biology

Figure 1. The schematic of IL-6_scfv-Notch-Gal4KRAB under low and high serum IL-6 level.

This device incorporates anti-IL-6 scFv (BBa_K4175000), the Notch core domain (BBa_K4175001), and ZF_GAl4_KRAB (BBa_K2446037) (Fig 1).

In this device, IL-6-scFv is a sensor for serum IL-6 level. The Notch domain undergoes two proteolytic cleavages upon the binding of ligands to the extracellular domain, which allows the release of the intracellular domain into the cytoplasm and the nucleus (Morsut et al., 2016). Gal4KRAB is a transcriptional inhibitor, where Gal4 binds to UAS (Gal4-binding region) in DNA and KRAB binds to the promoter attached to UAS, allowing inhibition of the downstream gene expression (Morsut et al., 2016; Witzgall et al., 1994).

IL-6 is found to play a major role in the pathophysiology of cytokine release syndrome (CRS), the most common adverse effect during CAR-T therapy (Shimabukuro-Vornhagen et al., 2018; Xiao et al., 2021). The elevated IL-6 during CAR-T therapy is mostly released by monocyte/macrophage cell lines induced by target cell pyroptosis upon T cell killing (Norelli et al., 2018; Shimabukuro-Vornhagen et al., 2018). Therefore, we created this device to set a negative ‘switch’ for CAR expression in the case of high serum IL-6 level. We hoped that this would help prevent severe CRS.

We expected that when the concentration of IL-6 is high, the binding of IL-6 with anti-IL-6 scFv would cause the Notch core domain to undergo proteolytic cleavages (Fig 1). As a result, the Gal4-KRAB domain would be released into the nucleus. We intended to express anti-CD19 CAR with UAS-pSV40 as the promoter in the CAR-T cells (see BBa_K4175012). Therefore, the DNA binding domain of Gal4 would bind to the UAS region, enabling the KRAB domain to inhibit the SV40 promoter. In this way, the expression of CAR would be inhibited. As the cytotoxicity effect of CAR-T cells would be weaker, the target cells would undergo less pyroptosis. Consequently, fewer monocytes/macrophages would be induced to release IL-6. The serum level of IL-6 would thus decrease.

When the concentration of IL-6 is low, IL-6R would fail to bind with IL-6 due to its low affinity. As the promoter upstream of CAR, pSV40, is a strong promoter, the CAR expression would be constitutive. Ideally, these would collectively allow the CAR-T cells to exert its cytotoxic effect under low IL-6 concentration (Fig 1).

Hopefully, these would help maintain the IL-6 level in a normal range during CAR-T therapy.

Characterization

Figure 2. The schematic map of MND-IL-6_scfv-Notch-Gal4KRAB-UAS-pSV40-aCD19CAR-P2A-mCherry plasmid.

IL-6_scfv-Notch-Gal4KRAB was synthesized by GenScript. Then the synthesized gene was cloned into MND plasmid together with UAS-pSV40-aCD19CAR-P2A-mCherry (BBa_K4175008) (Fig 2). 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. The expression of IL-6_scfv-Notch-Gal4KRAB and anti-CD19 CAR was confirmed using flow cytometry.

We then co-incubated IL-6_scfv-Notch-Gal4KRAB expressing CAR-T cells 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.

Unfortunately, we found that IL-6_scfv-Notch-Gal4KRAB failed to inhibit the cytotoxicity effect of CAR-T under high IL-6 level. This might suggest that the CAR expression was not suppressed.

Discussion

The major reason for the failure of IL-6_scfv-Notch-Gal4KRAB may lie in the property of Notch. It has been found that synthetic Notch (just like IL-6_scfv-Notch-Gal4KRAB) lacked the capacity of transducing signals when bound to soluble ligands. Instead, synthetic Notch worked well when encountering ligands that were bound to the cell membrane of another cell (Morsut et al., 2016). In our system, the ligand for the synthetic Notch receptor is IL-6, which is in its soluble form. This form of IL-6 may have no effect on Notch activation. Therefore, we concluded that synthetic Notch system was not appropriate for our purpose. This could be further proved by the failure of another synthetic Notch that we have created for the purpose, IL-6R-Notch-Gal4KRAB (BBa_K4175010). Instead, we created a new device (IL-6R-PD-1, BBa_K4175011) other than synthetic Notch as a negative ‘switch' for CAR activity. We found that IL-6R-PD-1 might be more useful in our context. For more details about IL-6R-PD-1, please visit BBa_K4175011.

References

Morsut, L., Roybal, K.T., Xiong, X., Gordley, R.M., Coyle, S.M., Thomson, M., Lim, W.A., 2016. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780–791. https://doi.org/10.1016/j.cell.2016.01.012

Norelli, M., Camisa, B., Barbiera, G., Falcone, L., Purevdorj, A., Genua, M., Sanvito, F., Ponzoni, M., Doglioni, C., Cristofori, P., Traversari, C., Bordignon, C., Ciceri, F., Ostuni, R., Bonini, C., Casucci, M., Bondanza, A., 2018. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat. Med. 24, 739–748. https://doi.org/10.1038/s41591-018-0036-4

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

Witzgall, R., O’Leary, E., Leaf, A., Onaldi, D., Bonventre, J.V., 1994. The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc. Natl. Acad. Sci. U. S. A. 91, 4514–4518. https://doi.org/10.1073/pnas.91.10.4514

Xiao, X., Huang, S., Chen, S., Wang, Y., Sun, Q., Xu, X., Li, Y., 2021. Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies. J. Exp. Clin. Cancer Res. CR 40, 367. https://doi.org/10.1186/s13046-021-02148-6