Part:BBa_K4175010
IL-6R-Notch-Gal4KRAB
Sequence and Features
- 10INCOMPATIBLE 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 1294
Illegal PstI site found at 1519
Illegal PstI site found at 1560
Illegal PstI site found at 1606
Illegal PstI site found at 2638 - 12INCOMPATIBLE 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 1294
Illegal PstI site found at 1519
Illegal PstI site found at 1560
Illegal PstI site found at 1606
Illegal PstI site found at 2638 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 60
Illegal BamHI site found at 76
Illegal BamHI site found at 1194
Illegal XhoI site found at 19 - 23INCOMPATIBLE 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 1294
Illegal PstI site found at 1519
Illegal PstI site found at 1560
Illegal PstI site found at 1606
Illegal PstI site found at 2638 - 25INCOMPATIBLE 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 1294
Illegal PstI site found at 1519
Illegal PstI site found at 1560
Illegal PstI site found at 1606
Illegal PstI site found at 2638
Illegal NgoMIV site found at 1432
Illegal NgoMIV site found at 2423
Illegal NgoMIV site found at 2558 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This device is comprised of extracellular human IL-6R (aa 1-309) (BBa_K4175013), the Notch core domain (BBa_K4175001), and ZF_GAl4_KRAB (BBa_K2446037) (Fig 1).
The usage and rationale of the synthetic device is quite similar to IL-6_scfv-Notch-Gal4KRAB (BBa_K4175008). Briefly speaking, we used IL-6R-Notch-Gal4KRAB as a negative ‘switch’ for CAR expression when the serum IL-6 level goes beyond normal. We hoped that implementation of this device to CAR-T cells would prevent severe cytokine release syndrome (CRS) from happening during CAR-T therapy, as IL-6 is a major culprit of CRS (Shimabukuro-Vornhagen et al., 2018). When the serum IL-6 level goes high, we expected that binding of IL-6 to IL-6R would trigger the proteolytic cleavage of Notch (Fig 1). Then the intracellular Gal4KRAB would be released into the nucleus. As we transfected the T cells with UAS-pSV40-aCD19-CAR (BBa_K4175008), the Gal4KRAB would then bind to the UAS region and inhibit pSV40 promoter. Consequently, the anti-CD19 CAR expression would be suppressed. The cytotoxic effect of CAR-T would be paused, and the monocytes/macrophages would temporarily stop producing more IL-6. Conversely, when the IL-6 level is low, IL-6R would not transduce signals due to its low affinity for IL-6 and the CAR would be constitutively expressed due to the strong promoter, SV40 (Fig 1). We hoped this would maintain the serum IL-6 level in a normal range.
The only difference between this part and Part:BBa_K4175008 lies in the IL-6 sensor. For BBa_K4175008, we used anti-IL6 scFv as the IL-6 sensor; while for this part, we used extracellular IL-6R as the sensor. These two sensors may have different affinities for IL-6, which means that the threshold for these two devices to exert their suppressive effect on CAR expression might be different.
For more details of usage and biology, please visit the part page of IL-6_scfv-Notch-Gal4KRAB (BBa_K4175008).
Characterization
IL-6R-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-6R-Notch-Gal4KRAB and anti-CD19 CAR was confirmed using flow cytometry.
We then co-incubated IL-6R-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-6R-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-6R-Notch-Gal4KRAB to suppress CAR expression may be that synthetic Notch receptors cannot respond to soluble ligands (Morsut et al., 2016). However, in our case, the ligand for IL-6R-Notch-Gal4KRAB is IL-6 in its soluble form. IL-6_scfv-Notch-Gal4KRAB (BBa_K4175008) also failed to exert its inhibitory effect, further proving that synthetic Notch system cannot respond well to soluble IL-6. 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
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
| biology | Human |