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.

Methods
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 proteins on target cells, transiently transfected cells were tested with primary antibody. Briefly, after one wash with FACS buffer (PBS + 2% BSA), cells were stained with primary antibodies for 40-60 min at room temperature (1:100 dilution, according to the manufacturer’s instructions). After one wash with FACS buffer cells were incubated with secondary antibody (Anti-mouse IgG Alexa Fluor™ 647) for 40-60 min at room temperature (1:2000 dilution). After two additional washes with FACS buffer (including 1 ug/mL DAPI to stain dead cells), cells were analyzed via flow cytometry for target expression.

FACS Co-culture Assays

CAR-expressing cells were co-cultured in a 1:1 ratio with target EGFR or CEA-expressing cells (HeLa WT, HeLa CEA+) or non EGFR or CEA-expressing cells (HeLa EGFR KO, HeLa WT) in RPMI medium for 18 hrs. Jurkat suspension cells were collected, washed in DPBS with EDTA 2mM and GFP fluorescence was assessed at FACSAria (BD) to quantify activation. In a similar fashion, Tmod-expressing cells were co-cultured in a 1:1 ratio with activator and blocker antigens expressing cells and GFP fluorescence was assessed to quantify blocking.  Median MFI and % activation and blocking was used to compare several conditions and optimize the best conditions for the assay.

Maximal activator enrichment was calculated from the flow data as %GFP(+) in the activated population to the right of the gate divided by %GFP(+) of the uninduced population to the right of the gate. Maximal blocker enrichment was calculated as %GFP(-) in the blocked population divided by %GFP(-) in the unblocked population.