Current Chimeric Antigen Receptor (CAR) T cell treatments can be profoundly successful in treating cancers, but there are challenges in developing new treatments to benefit more patients. These challenges relate to both safety and efficacy, often due to a lack of control over the strength and quality of engineered receptor signalling. We have developed a series of synthetic TM domains that adopt specific oligomeric states (monomers, dimers, trimers, tetramers), and when inserted into CARs to make programmable CARs (proCARs) they drive tuneable biochemical signalling and functional outputs.
While having one tuneable receptor signal in a cell is great, we are interested in developing two (or more) signalling receptors that can be independently tuned in the same cell. To measure the pairwise interactions of proCARs with different TM domains, we used FRET (Forster Resonance Energy Transfer), biochemical pull-downs, and a novel assay we call pull-sideways in live cells. The programmable oligomeric CARs interacted as expected (e.g. trimer with trimer, tetramer with tetramer), with minimal heterotypic interactions between the different oligomeric forms. We conclude that these synthetic TM domains can be combined together to make separate oligomers, giving us independently tuneable insulated signalling pathways.