De novo- designed transmembrane domains tune engineered receptor functions

How can the structural properties of transmembrane domains (TMDs) be precisely engineered to control the signaling and functional output of chimeric antigen receptors (CARs)? Current CAR designs often rely on natural TMDs, which can lead to unpredictable signaling due to cross-talk with endogenous T cell proteins.

The researchers developed a computational design strategy using the Rosetta software suite to create 'programmed membrane proteins' (proMPs). By incorporating membrane-solvation energy terms and a sequence diversification step that mimics natural TMD amino acid composition, they designed single-pass alpha-helical TMDs with defined oligomeric states (monomers, dimers, trimers, and tetramers). These designs were validated through bacterial selection systems, SDS-PAGE, and X-ray crystallography. The team then integrated these proMPs into CAR constructs to test their functional impact on mouse primary T cells.

The study demonstrates that CAR T cell activity, including cytokine release and in vivo antitumor potency, scales linearly with the oligomeric state of the engineered TMD. Specifically, the researchers found that their programmed CARs (proCARs) stimulated significantly lower inflammatory cytokine release compared to the commonly used CD28 TMD. They identified that the CD28 TMD promotes higher cytokine release by laterally recruiting endogenous costimulatory receptors, a side effect that the de novo-designed, orthogonal proMPs successfully avoided.

This work provides a powerful new toolkit for synthetic biology, enabling the rational design of receptors with predictable signaling properties. By decoupling the receptor's structural assembly from the native T cell machinery, researchers can better balance the antitumor efficacy of CAR T cells with the need to minimize toxicities like cytokine release syndrome (CRS), potentially leading to safer and more effective immunotherapies.