The Outpace Bio team developed a protein for CAR-T cells that simultaneously removes three regulators of T-cell exhaustion
The Outpace Bio team developed a protein for CAR-T cells that simultaneously removes three regulators of T-cell exhaustion
On August 14, a team from Outpace Bio and the University of Washington published a preprint on OUTLAST protein regulators. In experiments using human T-cells and mice implanted with human tumors, one of these regulators helped the cells remain active against the tumor for longer after repeated exposure.
CAR-T is a cell therapy in which immune T-cells are given a receptor that allows them to recognize a tumor. Within a tumor, these cells receive the same signal repeatedly. Over time, they become less effective at killing tumor cells and release fewer signaling molecules that immune cells use to coordinate their response.
Three related proteins take part in this internal program: NR4A1, NR4A2 and NR4A3. A 2019 study showed that simultaneously disabling all three genes in CAR-T cells improves their function more than disabling any one of them alone. The authors of the new preprint selected a region shared by all three NR4A proteins and designed a short binding protein called nr78.
When nr78 was fused to a fragment of the SPOP protein, the resulting construct directed bound NR4A proteins to the proteasome, the cellular machinery that breaks down proteins. A mutation at the binding site prevented the construct from binding and degrading NR4A and from sustaining CAR-T function. MLN4924, a compound that inhibits this intracellular degradation pathway, also prevented NR4A degradation. These experiments tested the complete mechanism: nr78 binds NR4A, and SPOP initiates their degradation.
The researchers first exposed CAR-T cells to H1975 lung cancer cells in three consecutive rounds. The SPOP-nr78 module helped the CAR-T cells continue killing tumor cells and releasing the signaling molecules IFN-γ and IL-2 for longer. In an experiment spanning eight such rounds, SPOP-nr78 preserved tumor-killing activity in cells from three donors. The variant with an altered NR4A binding site produced the same result as CAR-T cells without the regulator.
In mice implanted with H1975 tumor cells, the team compared three doses of CAR-T cells produced from the cells of two donors. Each dose group contained five animals. SPOP-nr78 restricted tumor growth even at a dose of 100 thousand CAR-T cells. All mice survived in every reported group that received cells containing this module. The authors also tested another version of the regulator in TCR-T cells, which are T-cells equipped with a receptor that recognizes the tumor protein PRAME. In this model, the cells also controlled the tumor more effectively.
This protein module can be encoded by a single short genetic insert: nr78 identifies a shared region in the three NR4A proteins, and SPOP initiates their degradation. In preclinical models, this approach helped two types of engineered T-cells maintain their antitumor activity for longer.