Reprogramming T cells in inhibitory tumor microenvironment with TGFβ redirectors for improved ultrasound controllable CAR-T therapy

Introduction:: Chimeric Antigen Receptor T-cell (CAR-T) therapy holds considerable potential in treating hematological malignancies, yet its efficacy in solid tumors is often compromised by an immunosuppressive tumor microenvironment (TME), characterized by factors such as Transforming Growth Factor-beta (TGFβ). Additionally, the phenomenon of "on-target off-tumor toxicity" presents a substantial challenge in CAR-T therapy. Recent advancements in focused ultrasound (FUS) controllable CAR-T cell technology offer the promise of controlled activation within specific tumor regions, thereby mitigating toxicity. However, the limited persistence of CAR molecules on T-cell surfaces within these inducible systems restricts their clinical utility. This study aims to circumvent these challenges through the innovative design of a series of customized synthetic TGFβ redirectors, which can be utilized to facilitate the modulation of inhibitory tumor microenvironments, reprogram inhibitory signaling into stimulatory signaling, and establish a synthetic signaling feedback loop to prolong the half-life of CAR molecules in T cells.


Materials and Methods:: A synthetic biology approach was adopted to construct the TGFβ redirectors. The design of TGFβ redirectors was initially optimized and validated in the NFKB or STAT signaling pathway reporter-expressing Jurkat T cell line by assessing various extracellular domain combinations and employing 2ng/ml soluble TGFβ treatment. Post-optimization, the function of TGFβ redirectors was examined in primary T cells from healthy donors. The phenotypic changes (IL2, IFNγ, TNFα expression) within the TME, with or without TGFβ redirectors, were assessed using functional assays and RNAseq after 2ng/ml soluble TGFβ treatment. Furthermore, TGFβ redirectors were utilized as signal amplifiers in FUS-CAR-T cells, ensuring prolonged CAR molecule presence. CAR molecule has been stained and quantified with AF647-Anti-Mouse IgG (H+L) Fab antibody at Day 1, 2 and 3 after CAR expression induction. The CAR-T cell efficacy have been validated in a mouse tumor model, with the caliber measurement and luciferase reading of tumor cells as readouts.


Results, Conclusions, and Discussions:: Our results showed that human protein-based TGFβ redirectors could successfully rewire T-cell function in the immunosuppressive TME upon TGFβ exposure. Different from the previous studies on TGFβ signaling pathway manipulation, this process achieved the customized transformation of inhibitory TGFβ signaling into endogenous stimulatory NFκB signaling or JAK/STAT signaling, and we managed to tailor immunotherapy for solid tumors by implementing different designs of TGFβ redirectors. Furthermore, a synthetic positive feedback loop using responsive elements corresponding to specific TGFβ redirectors has been developed to extend the CAR half-life on the T cell surface. Notably, the integration of an IL18 signaling motif with TGFβ redirectors exhibited remarkable efficacy in converting the inhibitory TGFβ signals into stimulatory signals, evident in the qPCR assay and RNA sequencing, and prolonged CAR expression up to 3 days compared to the 24-hour duration observed in the original inducible FUS-CAR design. This TGFβ redirector significantly enhanced the tumor elimination efficiency of FUS-responsive CAR-T cells in vivo. In summary, using a synthetic approach, we engineered a series of fully human transgene switches has been engineered to rewire T-cell function in an immunosuppressive microenvironment that enables the customized T-cell signaling in response to TGFβ and efficient tumor control of FUS controllable CAR-T cells. Given the wholly human protein-based nature of these TGFβ redirectors, they hold significant potential for clinical applications over other synthetic receptors. Future research will focus on refining this method by introducing TGFβ redirectors into the native TGFβ receptor site. This approach aims to accomplish the functional expression of TGFβ redirectors while simultaneously suppressing the inherent TGFβ signaling pathway in T cells. Furthermore, as TGFβ plays vital role in cellular development, we can foresee the broader applications of TGFβ redirectors in controlling cellular processes in other research areas.




Acknowledgements (Optional): : We gratefully acknowledge the financial support from National Institutes of Health (NIH), specifically through awards EB029122, GM140929, HL121365, HD107206, and CA262815.


References (Optional): : Trapani JA. The dual adverse effects of TGF-beta secretion on tumor progression. Cancer Cell. 2005 Nov;8(5):349-50. doi: 10.1016/j.ccr.2005.10.018. PMID: 16286241.

Wu Y et.al. Control of the activity of CAR-T cells within tumours via focused ultrasound. Nat Biomed Eng. 2021 Nov;5(11):1336-1347. doi: 10.1038/s41551-021-00779-w. Epub 2021 Aug 12. PMID: 34385696