High-throughput screen to identify and optimize NOT gate receptors for cell therapy

Logic-gated engineered cells are an emerging therapeutic modality that can take advantage of molecular profiles to focus medical interventions on specific tissues in the body.  However, the increased complexity of these engineered systems may pose a challenge for prediction and optimization of their behavior.  Here we describe the design and testing of a flow-cytometry-based screening system to rapidly select functional inhibitory receptors from a pooled library of candidate constructs. In proof-of-concept experiments, this approach identifies inhibitory receptors that can operate as NOT gates when paired with activating receptors.  The method may be used to generate large datasets to train machine-learning models to better predict and optimize the function of logic-gated cell therapeutics., Flow cytometry
 
To evaluate surface expression of CARs, cells were stained with soluble EGFR-Fc or CEA-fc followed by APC-labeled anti-human IgG Fc. Briefly, 0.25-0.5 ug/mL of protein was incubated for 40-60 min at RT (room temperature). Two additional washes with FACS buffer (PBS + 2% BSA), were then performed, followed by staining with streptavidin-fluorophore (fluorophore = PE or APC, as indicated) at 0.1 mg/mL at room temperature for 30 min. Washed twice with FACS buffer, cells were analyzed via flow cytometry for CAR expression (based on % of APC or PE-positive staining compared to unstained UTD cells, as indicated). To evaluate surface expression of the blocker, cells were labeled with biotinylated-pMHC probes, generated as described previously [16], tetramerized and prelabeled with streptavidin conjugated to an appropriate fluorochrome. After staining at 4C, median fluorescence intensity (MFI) was determined using a FACS Canto II flow cytometer. To evaluate surface expression of..., , # # High-throughput screen to identify and optimize NOT gate receptors for cell therapy

\[[https://doi.org/10.5061/dryad.np5hqc02v](https://doi.org/10.5061/dryad.np5hqc02v)]

Here we designed a flow-cytometry-based screening system to rapidly select functional inhibitory receptors from a pooled library of candidate constructs. This screen incorporates an NFAT-responsive reporter cell line to allow selection of reporter (+) and (-) cell populations by FACS after exposure to different stimuli. A library of blocker variants was constructed to test the concept of Tmod optimization and structure-activity inference from this cell-based functional screen. A Jurkat reporter clone was identified that maintained low background expression of green fluorescent protein (GFP) in the absence of a stimulus, and strong GFP induction by antigen. This Jurkat screening system for Tmod blockers was robust across multiple constructs, with signal:noise properties suggesting that enrichment of up to 15-fold...