Periodic Reporting for period 1 - T-FITNESS (FINE-TUNING T CELL NETWORKS OF EXHAUSTION BY SYNTHETIC SENSORS)
Periodo di rendicontazione: 2022-09-01 al 2023-08-31
Immunotherapies using tumor-fighting T cells, like TILs , or T cells engineered with specialized receptors (CAR or TCR), have been game-changers in cancer treatment. They have shown remarkable success in treating blood cancers like leukemia and lymphoma, but their efficacy against solid tumors has been underwhelming. A significant hurdle in making these therapies effective against solid tumors is what we call "T cell exhaustion”: during a protracted battle with tumor cells, T cells can become fatigued and are no longer able to function properly and to fight tumor cells efficiently. Our vision with T-FITNESS is to create exceptionally resilient T cells that can keep fighting and not get tired. To achieve this, we are developing molecular circuits that can lower the expression of the transcription factors (TFs) responsible for tiring out antitumor T cells. The challenge is that some of the TFs that make T cells exhausted are also important for their survival and effectiveness. Simply turning these TFs off completely is a bad idea because it would damage the T cells. Instead, we need to control these TFs very precisely to empower T cells to combat tumors effectively. Most current T-cell therapies use genetic tools to turn TFs on or off, but they can't control their expression very precisely. T-FITNESS sets itself apart by constructing a system that can adjust TF activity in T cells in a very fine-tuned way. We plan to use a genetic tool called CRISPR/Cas to insert synthetic "sensors" directly into well-defined sequences of the T cells’ DNA. These synthetic sensors are designed to detect specific signals, like a microRNA, that indicate when the T cells are becoming fatigued. As these sensors pick up these fatigue signals, they trigger precise adjustments in the T cell TFs to maintain their strength and effectiveness in the battle against cancer. T-FITNESS will seamlessly integrate into CAR-T, TCR-T, and TIL platforms, thereby unleashing the curative potential of T-cell therapy for an ever-growing number of cancer patients. This is particularly significant in Europe, where the number of new cancer cases per year is estimated to rise to 3.5 million by 2040.
The primary focus of the project's first year has been to uncover the microRNAs and TFs that control T cell exhaustion. We used a cell culture and a mouse model of T cell exhaustion to generate small RNA-sequencing and multi-omics datasets of functional and dysfunctional T cells. Through advanced bioinformatic analyses, we identified microRNAs that are consistently induced as T cells become exhausted in both settings. We confirmed and validated these findings through more sensitive analyses involving quantitative PCR. We have started applying this knowledge to design the microRNA-based exhaustion sensors, which will form the core of the T-FITNESS technology. We conducted a comprehensive analysis of the multi-omics data generated by T-FITNESS, combining it with publicly available datasets. Using the TETRAMER platform previously developed by CNRS (Cholley et al. Nat. Sys. Biol & App. 2018), we reconstructed the gene regulatory networks governing T cell exhaustion to pinpoint the TFs responsible for this process. This information will guide our selection of the "Exhaustion Master Regulator" TF to be targeted by the synthetic circuits developed by T-FITNESS. In the project's initial year, we also successfully developed and established a discovery pipeline for genome editing tools that will be employed to insert T-FITNESS sensors into T cell DNA. We employed a cell line as a model system and used a non-homologous end joining (NHEJ) assay as a proxy for determining the efficient generation of DNA double-strand breaks. We used this pipeline to identify high-performing reagents to insert microRNA sensors into untranslated regions of a representative exhaustion TF. We also began screening multiple small molecules to improve the knock-in efficiency in human T cells and started optimizing the electroporation parameters in small-scale experiments using Miltenyi’s CliniMACS Prodigy Electroporation system. Finally, within the framework of our cell and gene therapy (CGT) subportfolio activities, we forged a collaborative partnership with project XPAND. Our shared objective is to enhance the CRISPR/Cas gene editing platform for T cells and hematopoietic cells. Planned activities will be initiated in 2024.
Apart from a limited number of studies that have focused on the pro-exhaustion activities of specific microRNAs (e.g. miR-31 and miR-155), and a recent study outlining the microRNA signature of exhausted T cells during chronic infection (Stelekati et al. PNAS 2022), our understanding of the microRNA profile of T cells experiencing exhaustion in tumor-bearing hosts is significantly lacking. By studying human T cells in an animal model of CAR T cell exhaustion, we have achieved a significant milestone. We have created a comprehensive microRNA map of both functional and exhausted T cells. This map will not only provide essential guidance for the development of T-FITNESS synthetic sensors but will also serve as a key reference for designing other therapeutic approaches targeting exhaustion through microRNA modulation. Transcriptomic and epigenetic profiles of exhausted T cells have been extensively characterized in the setting of chronic viral infections and in TILs but less is known about the exhaustion programs affecting CAR T cells. Overall, our effort to analyze several publicly available datasets together with those generated in our in vivo model of CAR T cell exhaustion has led to the identification of highly conserved exhaustion TFs. Notably, among the top-ranked 35 TFs, 22 of them match the "Universal TF" list outlined by Zheng et al. (Science 2021), while 13 represent TFs not previously associated with exhaustion. These newfound factors provide potential novel therapeutic targets for interventions like T-FITNESS or other approaches. The development phase of T-FITNESS technology is in its initial stages. Further research within the project scope will yield an optimized design of the synthetic circuits and evaluate their impact on T cell antitumor efficacy.