A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

Significance Molecular ON-switches are important tools in chemical biology, enabling protein–protein interactions to be regulated by small molecules. However, currently available ON-switches that induce conditional heterodimerization are suboptimal for therapeutic applications. In this study, we present an ON-switch system based on human retinol binding protein 4 (hRBP4) and the orally available small molecule A1120. Two distinct protein scaffolds, FN3 and rcSso7d, were successfully engineered to bind to hRBP4 in a small molecule-dependent manner, demonstrating the flexibility of the system. The binders specifically associated with the drug-induced conformation of hRBP4. Our study demonstrates that lipocalin-based ON-switches can enable specific regulation of protein heterodimerization and provides proof of concept for potential applications in controlling the activity of human CAR T cells.


Screening for binders using yeast display
Yeast-displayed libraries based on rcSso7d (libraries rcSso7d-11 and rcSso7d-18, library size of 1.4 × 10 9 each(1)) and the library G4 based on the FN3 domain(2) with a library size of 2.5 × 10 8 were used. S. cerevisiae (strain EBY100) was grown in SD-CAA medium (20 g/L glucose, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 11.85 g/L sodium citrate dihydrate and 7.4 g/L citric acid monohydrate) at 30°C overnight while shaking. Cells were passaged to an OD600 of 1, followed by incubation at 30°C while shaking. When an OD600 of 4 was reached, cells were centrifuged and resuspended to an OD600 of 1 in SG-CAA medium (2 g/L glucose, 20 g/L galactose, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 10,2 g/L disodium hydrogen phosphate and 4,82 g/L sodium phosphate monobasic).
Cells were incubated at 20°C overnight while shaking and subsequently harvested by centrifugation. Two cycles of bead selection were performed with magnetic streptavidincoated Dynabeads (Life Technologies) loaded with biotinylated hRBP4 as described previously (2)(3)(4). To avoid the selection of non-specific binders or binders which are specific for streptavidin, negative selections (with bare beads) were performed between cycles of positive selection. In all bead selections, A1120 (Sigma-Aldrich) was present at a concentration of 5 µM. After the second bead-selection cycle, error prone PCR (epPCR) was conducted in order to introduce additional mutations into rcSso7d and FN3 genes. The template DNA was subjected to 19 PCR cycles using nucleotide analogs ( for the FN3-based binders, respectively. The resulting product was used as a template for further amplification by a second PCR using the same primers that were also used for epPCR. Afterwards, EBY100 cells were electroporated with linearized pCTCON2 vector (3,5) and insert DNA (PCR product) as described previously (6). After a third round In positive selections (i.e. selection for hRBP4 binding) A1120 was added to a final concentration of 5 µM, whereas in negative selections (i.e. selection for non-binding to hRBP4) no A1120 was present. Cells were incubated with varying concentrations of biotinylated hRBP4 (depending on the selection round) and 5 µg/mL mouse anti-c-myc and diafiltration was performed with the diafiltration TFF system (Merck Millipore) using Pellicon XL membranes (5 kDa cut-off, Merck Millipore) to exchange the medium with loading buffer (50 mM sodium phosphate, 500 mM NaCl and 5 mM imidazole, pH 7.4).
Briefly, the diafiltrated supernatant was applied to a HisTrap FF column (GE healthcare) connected to an ÄKTA HPLC purifier system (GE healthcare). Elution was performed using a linear imidazole gradient (from 5 to 500 mM imidazole in a 50 mM sodium phosphate buffer, pH 7.4, containing 500 mM NaCl). Fractions containing hRBP4 were pooled and concentrated using Amicon Ultra-15 10K centrifugal filters (Merck Millipore).

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Buffer was exchanged to phosphate buffered saline (PBS, pH 7.4). Before preparative size exclusion chromatography (SEC), a fraction of RBP4 was labelled with biotin using the EZ-Link Sulfo-NHS-LC-LC-Biotin kit (Thermofisher Scientifc). Subsequently, preparative SEC was performed using a Superdex 200 column (10 mm x 300 mm, GE Healthcare) to remove remaining unbound biotin molecules.

Soluble expression of selected hRBP4-binders
After

Size exclusion chromatography
For analytical SEC analysis, the respective binder in running buffer (PBS supplemented with 200 mM NaCl) was filtered through a 0.1 µm Ultrafree-MC filter (Merck Millipore).
Subsequently, 25 µg protein were applied to a Superdex 200 column (10 mm x 300 mm, GE Healthcare) connected to an HPLC Prominence LC20 system (Shimadzu) and eluted with a flow rate of 0.75 mL/min at 25°C.

Expression and purification of hRBP4 with cleavable His6-Tag for crystallization
For crystallization, hRBP4 was cloned into the pPICZα A vector with an additional cleavage site after the His6-tag for recognition by the human rhinovirus 3C (HRV 3C) protease in order to produce protein without a tag. The P.pastoris strain X-33 was transformed with the sequence-verified plasmid and the protein was expressed and purified as described above. After purification on a His Trap column (GE Healthcare), the protein was incubated with HRV 3C protease (fused to a glutathione-S-transferase tag) for cleavage of the His6 tag (digestion overnight at 4°C with ten-fold excess of HRV 3C protease). Finally, the digested sample was loaded onto a GSTrap HP column (GE Healthcare) to retain the protease and onto a His Trap column (to remove the cleaved tag), followed by a final purification by preparative SEC using a Superdex 200 column (10 mm x 300 mm, GE Healthcare).

Crystallization of the hRBP4-A1120-RS3 complex
Purified hRBP4 in PBS was mixed with 1.5-fold molar excess of RS3 in PBS and 500 µM  were incubated for 20 h at 37°C before they were used for further experiments.

Flow cytometric analysis of CAR expression
Cells were counted using Accucheck counting beads (Life Technologies)      24 Table S1.