Part:BBa_K3132104
UAS_MinimalCMV_CAR_IL-2_His tag
This composite part consists of one of our synthetic promoters, UAS_minimalCMV(BBa_K3132100), and downstream gene in our first layer cell, CAR_IL-2. The corresponding SynTF of UAS_minimalCMV is Gal4-VP64 (BBa_K3132000), which can bind to the UAS(4*Gal4 binding sites) and activate the minimalCMV promoter, therefore initiating the expression of the downstream gene. As for the design of CAR, the second generation CAR design, which fused a scFv, a CD8a hinge, a CD8 transmembrane domain, a 4-1BB intracellular domain and a CD3ζ chain in tandem was used in our project (BBa_K3132016) (Fig. 1).We designed two chimeric receptors that contain scFv derived from antibodies that recognize human EGFR and HER2, two tumor surface antigens we chose to target. Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) were chosen considering their safety as clinical targeted drugs. We inset the gene of rIL-2_His tag behind CAR. In this way, after Gal4-VP64 binds to the UAS_minimalCMV promoter, the expression of CAR and IL-2-tag will be activated. CAR will be expressed on the cell surface and directly recognizes the EGFR and HER2 on the tumor cell surface, performing the killing function; while the secretion of IL-2-tag will be induced, serving as an indicator of the medical conditions and a signal molecule to trigger the response of external device cells. Constitutively expressed blue fluorescent protein (BFP) was placed downstream of the inducible CAR transgene to identify transduced NK-92 cells (Fig. 1), which is under the control of a PGK promoter. With the fluorescence intensity we can verify the levels of co-transduction.
Design and Construction of Core Synthetic Device Based on Immune Cells:
After verifying the killing activity of CAR NK cells, we next set out to construct the core cells of our Theranostic nanobots. Instead of directly using CARs to recognize antigens, we let the NK-92 cells first express synNotch receptors to identify antigens, then leading to the expression of CARs and other genes. It is because the reporters for the activation of CAR is not stable. We constructed two lentiviral vectors for co-transfection of either anti-EGFR or anti-HER2 core cells (Fig. 1a and Fig. 1b). As for anti-EGFR core cell, the first vector encodes an EGFR-specific synNotch receptor bearing a Gal4-VP64 intracellular transcription activation domain (Fig. 1a). A scFv derived from the anti-EGFR antibody matuzumab was used in the synNotch receptor as it has a distinct epitope compared with that of cetuximab. The second vector we generated contained a fusion gene encoding the same EGFR-CAR and a His-tagged IL2 gene, under the control of the upstream activating sequence (UAS) promoter that is activated by Gal4-VP64 released after engagement of the synNotch receptor. The two coding sequences were linked together using 2A sequence peptides. Similar design was adopted for the HER2-antigen, we used an anti-HER2-domianⅠantibody H2-18 for the synNotch receptor, which has a far distinct epitope compared with that of trastuzumab for TTZ.CAR. In the second vector, a FLAG tagged IL-2 is utilized to report the role of the cell (Fig. 1b). NK-92 cells were co-transduced with both lentiviral vectors and co-transduction was verified by EGFR-Fc protein and BFP expression (Fig. 1c). Double positive NK-92 cells were enriched by fluorescence-activated cell sorting, and only about 10% of NK-92 cells were αEGFR+BFP+ and carried the full gene circuits. The double positive cells were termed as MTZ-synNotch.IC9.UAS-CTX.I2his cells. Similar methods were used to sort the TTZ-synNotch.IC9.UAS-CTX.I2FLAG cells.
Killing activity and Signal Molecules Secretion of Core Synthetic Devices
We next cultured sorted synNotch NK cells, and CAR-NK cells with MCF-7 EGFR, MCF-7 HER2 to test recognition of different antigen expressing tumor cells(Fig. 2a). Both CTZ.CAR-NK92 cells and TTZ.CAR-NK92 cells effectively killed EGFR+ or HER2+ cancer cells in 4 and 24 h co-cultures. By contrast, synNotch NK-92 cells recognized cancer cells expressing EGFR or HER2, and comparable lytic activity to CAR-NK-92 cells required 24 h of co-culture, consistent with previous reports showing that 12–24 h is required to fully upregulate CAR expression after engagement of the synNotch receptor1,2. In addition, the presence of antigen was sufficient to induce secretion of the IL-2-Tag protein at 24h, which is an indicator of the medical conditions and is a signal molecule to trigger the response of external device cells(Figure 2b).
References
[1] Roybal, K. et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 167, 419-432.e416 (2016).
[2] Srivastava, S. et al. Logic-Gated ROR1 Chimeric Antigen Receptor Expression Rescues T Cell-Mediated Toxicity to Normal Tissues and Enables Selective Tumor Targeting. Cancer cell 35, 489-503 e488, doi:10.1016/j.ccell.2019.02.003 (2019).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 395
Illegal NotI site found at 632 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 271
Illegal NgoMIV site found at 340 - 1000COMPATIBLE WITH RFC[1000]
| None |