De novo-designed transmembrane domains tune engineered receptor functions

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• De novo-designed receptor transmembrane domains (TMDs) offer precise control over cellular receptor functions.
• Programmed membrane proteins (proMPs) are single-pass α-helical TMDs designed for self-assembly via defined interfaces.

Purpose of the Study:

• To develop a de novo design strategy for generating proMPs.
• To program specific oligomeric interactions into chimeric antigen receptors (CARs).
• To investigate the impact of engineered TMDs on CAR T cell activity and antitumor function.

Main Methods:

• Utilized a de novo design strategy to create proMPs with defined self-assembly interfaces.
• Engineered proMPs into CARs and expressed them in mouse primary T cells.
• Assessed in vitro CAR T cell cytokine release and in vivo antitumor activity across varying oligomeric states (monomers to tetramers).
• Compared the activity of engineered CARs with those using the CD28 TMD.

Main Results:

• CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the encoded oligomeric state of the engineered TMDs.
• All programmed CARs exhibited lower T cell cytokine release compared to the CD28 TMD.
• The CD28 TMD was shown to elevate cytokine release via recruitment of the endogenous CD28 costimulatory receptor.

Conclusions:

• Precise de novo design of orthogonal and modular TMDs enables predictable programming of receptor structure.
• This approach allows for predictable tuning of CAR T cell activity for synthetic biology applications.
• Engineered TMDs offer a novel method for controlling CAR T cell effector functions and therapeutic efficacy.