Trimeric Redesign of a CAR-T Costimulatory Transmembrane Domain

Background

Chimeric antigen receptor (CAR) T cells rely on costimulatory domains to sustain activation, and 4-1BB is among the most widely used. In its native context the receptor assembles as a trimer, but conventional CAR constructs place its transmembrane domain (TMD) in an architecture that does not preserve that geometry.

Building on the host lab’s proCAR3 design hypothesis, this project asks a direct question: if the 4-1BBζ TMD is redesigned back into its natural trimeric form, does T cell activation improve?

Method

The work runs on two tracks.

Dry lab — computational screening. I independently ran 2 µs molecular dynamics simulations across 4 candidate designs to test whether each assembles into a stable trimer, using GROMACS with ΔG prediction, Rosetta, and ESM2 to guide and evaluate the designs.

Wet lab — functional validation. Collaborating with the team, I helped optimise 4 TMD variants, then ran flow cytometry and degranulation assays against 2 tumour cell lines, confirming surface expression before measuring function.

Techniques: flow cytometry, degranulation assay, HEK293T culture, GROMACS, ΔG prediction, Rosetta, ESM2.

Results

• Surface expression of 73–74% was confirmed for the TMD variants across both tumour cell lines, along with T cell activation — the redesigned constructs traffic correctly and work.
• 85%+ contact retention across the MD screen, confirming the redesigned TMD assembles into a stable trimer and holds that form.

Work is ongoing, with functional comparison against the conventional 4-1BBζ architecture as the current focus.

Fully funded through the WEHI InSPIRE program.