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We set out to evaluate whether our anti-CD19 SynNotch receptor could activate the TRE3GS-EGFP reporter in HEK293T in a culture assay with CD19 ligand. All conditions were normalised to the fully induced state (S+R+CD19 = 100%). Bars in the graph represent mean normalised GFP and whiskers indicate the 95% confidence interval.
Figure 1: SynNotch Reporter Activation
Stimulation assay of SynNotch+Reporter cells with CD19 ligand yielded the highest GFP signal, confirming that the receptor is functional and responsive to its cognate antigen.
Cells expressing SynNotch and the reporter but without CD19 still displayed roughly two-thirds of the fully induced signal, while reporter-only cells stimulated with CD19 gave about one-quarter. Together, these controls reveal a measurable basal ("OFF-state") signal from a combination of ligand-independent SynNotch cleavage and background fluorescence in the assay.
Introducing our tetracycline-mimic inhibitor peptide under different promoters shifted the response downward in a graded fashion: CMV_TIP reduced output to ~62%, CAG_TIP to ~52%, and EF1α_TIP to ~47% of the fully induced signal.
This pattern is consistent with the inhibitor raising the activation threshold as designed. EF1α produced the most consistent reduction, while CAG showed more variability between replicates.
These findings demonstrate that the engineered inhibitor can tune SynNotch activation in a promoter-dependent manner. Even at this early stage, EF1α appears to balance expression strength and stability, making it a promising candidate for further cycles.
At the same time, the high background in SynNotch+Reporter cells without CD19 ligand indicates that the system still needs further leak suppression or improved assay conditions to achieve a true OFF state.
We set out to test whether increasing the EF1α-driven inhibitor (TIP) would lower SynNotch output in a graded manner. To reduce baseline leak, we fixed SynNotch:Reporter at 1:10 and titrated TIP at four doses (S:R:E = 1:10:2, 1:10:5, 1:10:10, 1:10:20). The assay and readout matched our earlier setup, with lysate GFP measured at the 40-hour endpoint.
Figure 2: EF1α-TIP Dose-Response Analysis
In practice, the SynNotch plasmid volume became too low under this configuration. As a result, SynNotch expression fell below the functional threshold. Both the positive control (SynNotch+Reporter with CD19) and the negative control (SynNotch+Reporter without CD19) produced similarly low signals.
The four inhibitor doses were indistinguishable within variance, and no monotonic trend emerged. This outcome indicates that the experiment became SynNotch-limited rather than inhibitor-limited.
With too little receptor on the membrane, even robust CD19 stimulation could not produce a measurable difference above background. Any real effect of TIP dosing was therefore masked, and the dataset could not support dose-response fitting or an IC₅₀ estimate.
We will repeat the study with SynNotch restored above its activation threshold while keeping Reporter at 1:10. A modest increase in S (for example S:R = 1:8 or 1:5) will be paired with the same TIP doses and a constant total DNA per well.
Parallel verification of SynNotch expression will confirm that receptor abundance is no longer limiting. Under these conditions, we expect the EF1α-TIP series to reveal a clear, graded suppression of the reporter.