Periodic Reporting for period 2 - T-FITNESS (FINE-TUNING T CELL NETWORKS OF EXHAUSTION BY SYNTHETIC SENSORS)
Reporting period: 2023-09-01 to 2025-02-28
Immunotherapies using tumor-targeting T cells—e.g. TILs or T cells engineered with CARs or TCRs—have revolutionized cancer treatment, particularly for blood cancers like leukemia and lymphoma. However, their success against solid tumors has been limited. A major barrier is T cell exhaustion: during prolonged engagement with tumor cells, T cells lose their function and become ineffective. T-FITNESS aims to overcome this by creating T cells that remain resilient and functional over time. Our approach focuses on developing synthetic molecular circuits that finely regulate the expression of exhaustion-associated transcription factors (TFs). These TFs are double-edged swords: while they contribute to T cell dysfunction, they are also essential for cell survival and activity. Simply turning them off isn’t viable—it risks weakening or killing the T cells. Instead, T-FITNESS offers a precision-control strategy, adjusting TF activity in a responsive, context-aware manner. Most current therapies use static genetic switches that lack fine control. In contrast T-FITNESS uses CRISPR/Cas-mediated insertion of synthetic "sensors" into precise locations in the T cell genome. These sensors detect intracellular signals—such as specific microRNAs—that indicate when a T cell is becoming exhausted. In response, the system dynamically adjusts TF activity, helping the T cell recover and continue attacking the tumor. This technology is designed to integrate seamlessly into CAR-T, TCR-T and TIL platforms, extending the durability and effectiveness of T-cell therapies. As cancer incidence continues to rise—projected to reach 3.5 million new cases annually in Europe by 2040—T-FITNESS offers a promising path to make cell therapies more effective for a broader range of patients, especially those with solid tumors.
T-FITNESS began by exploring the molecular mechanisms driving T cell exhaustion. By generating and analyzing small RNA-sequencing data from both in vivo and in vitro models, the consortium identified and validated a set of microRNAs consistently associated with exhaustion. These findings enabled the development of synthetic gene circuits that respond to intracellular exhaustion signals. Simultaneously, the project prioritized key transcription factors involved in the regulation of exhausted T cells through integrative bioinformatic analysis of public and in-house datasets. A user-friendly tool for single-cell regulatory network analysis was also developed and is now in its final stages. These insights informed the design of miRNA-responsive synthetic circuits, which demonstrated robust and tunable control of gene expression in primary human T cells. To advance toward clinical application, T-FITNESS developed optimized genome editing protocols, achieving high-efficiency knock-ins with minimal impact on cell viability. Strategies included donor template optimization, repair pathway modulation, and the use of high-fidelity editing tools, all of which enhanced editing precision and performance. Functional studies confirmed that circuit-equipped T cells exhibited reduced exhaustion, improved proliferation, and sustained effector function under chronic stimulation in vitro. To advance T-FITNESS technology to the clinic, GMP-compliant manufacturing workflows were established using automated platforms. These workflows achieved high editing efficiency, scalability, and product quality, making them suitable for therapeutic development. With support from the Hop-On facility grant, the project also initiated the development of protein-based inhibitors targeting exhaustion-related transcription factors. In parallel, additional miRNA-responsive systems were created, offering new strategies for context-sensitive gene regulation. Finally, a collaboration with project XPAND was established to co-develop enhanced genome editing platforms for T cells and hematopoietic cells, with activities set to begin in 2025.
T-FITNESS has achieved major advances beyond the current state of the art in understanding T cell exhaustion and engineering responsive T cell therapies. Biologically, the project generated the first high-resolution microRNA profile distinguishing functional from exhausted human T cells in a CAR T cell model. This fills a critical gap in the field and provides a foundation for designing exhaustion-sensing synthetic circuits. T-FITNESS also identified many transcription factors enriched in exhausted T cells, including novel candidates not previously linked to exhaustion. These findings open new avenues for therapeutic modulation. On the engineering front, T-FITNESS developed miRNA-responsive gene circuits capable of adjusting gene expression based on exhaustion signals. These circuits demonstrated consistent performance in human T cells, enabling precise control without compromising viability. The project also optimized CRISPR-based genome editing, achieving up to 90% knock-in efficiency and high specificity. Functionally, engineered T cells showed reduced exhaustion, improved proliferation, and sustained tumor-killing capacity under repeated stimulation in vitro. These technologies were integrated into GMP-compliant manufacturing workflows using the CliniMACS Prodigy platform, outperforming standard methods in efficiency and scalability. AAV6-based delivery further enhanced clinical readiness. Additionally, the project launched a new line of work on protein-based inhibition of exhaustion-driving transcription factors, identifying promising AI-designed inhibitors. Complementary miRNA-responsive protein-based systems for gene regulation were also developed, expanding the platform’s capabilities.Together these advances establish T-FITNESS as a leading platform for next-generation T cell therapies, combining biological insight, precision control, and clinical scalability to enhance cancer immunotherapy.