Rational Design of Next-Generation Engineered T Cells for Enhanced Cancer Immunotherapy

A major challenge for engineered T cells to combat cancer is their limited persistence. The goal of this project is to enhance the persistence of T cells by harnessing 4-1BB using structure-informed protein design. As a co- stimulatory molecule, 4-1BB improves T cell long-term immune responses. Incorporating the 4-1BB signaling domain into chimeric antigen receptors (4-1BB CAR) significantly enhances the therapeutic effects in treating hematological malignancies. However, the scope and the long-term efficacy of 4-1BB CAR-T treatment remains limited, especially for patients with solid tumors, highlighting the need for a fundamental shift in the 4-1BB CAR protein design. 4-1BB (CD137/TNFRSF9) belongs to the tumor necrosis factor receptor (TNFR) superfamily. Protein structural studies have shown that effective TNFR signaling relies on specific high-order receptor clustering. However, current protein engineering approaches for harnessing 4-1BB have overlooked this crucial requirement. Thus, the 4-1BB signals initiated by current 4-1BB CAR design does not involve TNFR like-receptor clustering. Based on the importance of receptor clustering in native TNFR signaling amplification, we postulated that the absence of this critical arrangement of 4-1BB CAR results in inefficient 4-1BB signaling. Our preliminary experiment showed that the efficacy of CAR-resident 4-1BB is limited compared to endogenous 4-1BB activation in supporting CAR-T persistence. We hypothesize that high-order receptor clustering is needed for a synthetic 4-1BB to effectively support CAR-T cell activity. We have two specific aims. Aim 1 is to identify novel key factors required for robust 4-1BB signaling by cross-examining how these two pathways diverge (CAR-resident 4-1BB v.s. endogenous 4-1BB). Aim 2 is to employ structure-guided protein design to create a potent synthetic 4-1BB capable of forming TNFR-like clustering without the need for 4-1BB ligand. Cancer cells express limited 41BB ligand to evade immune surveillance. This engineered approach will deliver potent 4-1BB signals to enhance the persistence of engineered T cells when 4-1BB ligand is lacking. The success of the project will fundamentally advance our knowledge for modulating 4-1BB pathways to improve T cell persistence and open the door to enhance the durability of various types of engineered T cells beyond CARs. Collectively, our project will mark a leap forward in protein engineering for immunotherapy. Project Number: 1R21CA307944-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Ju Fang Chang | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $399,829 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 CDPT-Y (56)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11281089