Periodic Reporting for period 1 - SynT (Generation, validation and use of a synthetic reporter of CAR T cell products function and dysfunction)
Reporting period: 2024-03-01 to 2025-08-31
CAR T-cell therapies have transformed the treatment of certain blood cancers, demonstrating that a patient’s own immune cells can be re-engineered to eliminate tumours. Despite these breakthroughs, their use remains limited.
The ERC Proof of Concept project SynT set out to address a fundamental limitation in the development of CAR T-cell therapies: the lack of tools to monitor, in real time and under conditions relevant to product manufacturing and treatment, whether engineered immune cells remain functional or begin to drift into dysfunctional or exhausted states.
The Problem:
Current CAR T-cell engineering and testing rely on indirect or late-stage readouts that do not capture the dynamic changes T cells undergo during activation, stress, or exhaustion. This makes it difficult to predict how reliably a given CAR T-cell product will perform and hampers efforts to design improved versions or identify interventions that stabilise T-cell fitness. These limitations contribute to inefficiencies in the development pipeline and to the high costs and uncertain outcomes that characterise the CAR T sector.
The Solution:
SynT aims to create a set of synthetic genetic reporters that can indicate when a CAR T cell is in a functional state or has transitioned toward dysfunction. These reporters are based on synthetic locus control regions (sLCRs), which act as DNA “switches” capable of sensing and reflecting specific T-cell states. The concept underpinning SynT is that such reporters could enable real-time monitoring of engineered T cells and support better identification of factors that influence their fitness. This, in turn, would guide the development of more robust next-generation immune-cell therapies.
Innovative Approach:
SynT combines computational design with synthetic biology to develop regulatory DNA elements that respond to molecular signatures of T-cell function or dysfunction. By demonstrating the feasibility of designing such synthetic regulatory logic, SynT highlights a new strategy for improving the predictability and performance of future immunotherapies.
The ERC Proof of Concept project SynT set out to address a fundamental limitation in the development of CAR T-cell therapies: the lack of tools to monitor, in real time and under conditions relevant to product manufacturing and treatment, whether engineered immune cells remain functional or begin to drift into dysfunctional or exhausted states.
The Problem:
Current CAR T-cell engineering and testing rely on indirect or late-stage readouts that do not capture the dynamic changes T cells undergo during activation, stress, or exhaustion. This makes it difficult to predict how reliably a given CAR T-cell product will perform and hampers efforts to design improved versions or identify interventions that stabilise T-cell fitness. These limitations contribute to inefficiencies in the development pipeline and to the high costs and uncertain outcomes that characterise the CAR T sector.
The Solution:
SynT aims to create a set of synthetic genetic reporters that can indicate when a CAR T cell is in a functional state or has transitioned toward dysfunction. These reporters are based on synthetic locus control regions (sLCRs), which act as DNA “switches” capable of sensing and reflecting specific T-cell states. The concept underpinning SynT is that such reporters could enable real-time monitoring of engineered T cells and support better identification of factors that influence their fitness. This, in turn, would guide the development of more robust next-generation immune-cell therapies.
Innovative Approach:
SynT combines computational design with synthetic biology to develop regulatory DNA elements that respond to molecular signatures of T-cell function or dysfunction. By demonstrating the feasibility of designing such synthetic regulatory logic, SynT highlights a new strategy for improving the predictability and performance of future immunotherapies.
Work performed and main achievements
During the SynT Proof of Concept project, our team achieved a series of key milestones in the development, optimisation, and strategic positioning of a synthetic cis-regulatory DNA in engineered T cell products development. These achievements establish a solid foundation for future translational and collaborative work in oncology and now extend this to autoimmunity.
1. Enhancement of Computational Design Tools
Insights gained during experimental testing informed improvements to our synthetic DNA design platform. A next-generation version of the LSD design suite (LSD+) was assembled, incorporating updated logic for the selection and optimisation of regulatory DNA elements. This software provides enhanced capabilities for future reporter development efforts.
2. Development of Synthetic Reporters for T-Cell Function and Dysfunction
Following the pilot work, using the LSD+ suite, we successfully designed, constructed, and implemented four synthetic locus control region (sLCR)-based reporters reflecting functional activation states and capturing dysfunctional or exhausted states. These reporters constitute the core technological output of SynT and provide the basis for more informative monitoring of engineered immune-cell behaviour.
3. Integration of Reporters into CAR T Cells
Using established CAR architectures, the synthetic reporters were co-transduced into CD4+ T cells together alongside CARs directed against clinically relevant targets. This integration enabled controlled evaluation of reporter activity and laid the groundwork for future applications requiring dynamic readouts of T-cell state.
4. Characterisation Under Defined Experimental Conditions
The reporter cells were analysed under standardised stimulation conditions using flow cytometry and molecular assays. These assays enabled us to test biologically meaningful activation and differentiation cues, a key requisite to assess the feasibility of sLCR-based indicators for monitoring CAR T-cell state transitions.
5. Establishment of New Collaborative Partnerships
To further develop and validate the SynT reporters, formal collaboration agreements were signed with two external institutions specialising in T-cell engineering and validation. These partnerships extend the reach and sustainability of the technology beyond the ERC funding period.
6. Strategic Redirection Toward Autoimmunity Applications
During the project period, major shifts in the CAR T-cell therapy commercial landscape underscored the need for repositioning our long-term strategies. In response, SynT expanded its intended application space toward autoimmune diseases, where selectivity and temporal activation are key. This strategic repositioning broadens the impact and relevance of the SynT technology.
Future Outlook
Building on the SynT proof of concept, future work will focus on refining and extending the reporters within collaborative academic networks and, where appropriate, in structured translational programmes. The broader potential of SynT will continue to be explored through the partnerships and consortium efforts established during the project period, and to align our future commercial strategy with the current drastic shift in the cell therapy industry.
Conclusion
SynT has delivered a comprehensive proof of concept for the use of synthetic regulatory DNA elements to monitor CAR T-cell functional states. The project has strengthened computational tools, generated the intended technological assets, and positioned the technology for continued development across oncology and autoimmunity. These achievements advance the broader goal of creating more precise, predictable, and adaptable engineered immune-cell therapies despite the substantial market pressures.
During the SynT Proof of Concept project, our team achieved a series of key milestones in the development, optimisation, and strategic positioning of a synthetic cis-regulatory DNA in engineered T cell products development. These achievements establish a solid foundation for future translational and collaborative work in oncology and now extend this to autoimmunity.
1. Enhancement of Computational Design Tools
Insights gained during experimental testing informed improvements to our synthetic DNA design platform. A next-generation version of the LSD design suite (LSD+) was assembled, incorporating updated logic for the selection and optimisation of regulatory DNA elements. This software provides enhanced capabilities for future reporter development efforts.
2. Development of Synthetic Reporters for T-Cell Function and Dysfunction
Following the pilot work, using the LSD+ suite, we successfully designed, constructed, and implemented four synthetic locus control region (sLCR)-based reporters reflecting functional activation states and capturing dysfunctional or exhausted states. These reporters constitute the core technological output of SynT and provide the basis for more informative monitoring of engineered immune-cell behaviour.
3. Integration of Reporters into CAR T Cells
Using established CAR architectures, the synthetic reporters were co-transduced into CD4+ T cells together alongside CARs directed against clinically relevant targets. This integration enabled controlled evaluation of reporter activity and laid the groundwork for future applications requiring dynamic readouts of T-cell state.
4. Characterisation Under Defined Experimental Conditions
The reporter cells were analysed under standardised stimulation conditions using flow cytometry and molecular assays. These assays enabled us to test biologically meaningful activation and differentiation cues, a key requisite to assess the feasibility of sLCR-based indicators for monitoring CAR T-cell state transitions.
5. Establishment of New Collaborative Partnerships
To further develop and validate the SynT reporters, formal collaboration agreements were signed with two external institutions specialising in T-cell engineering and validation. These partnerships extend the reach and sustainability of the technology beyond the ERC funding period.
6. Strategic Redirection Toward Autoimmunity Applications
During the project period, major shifts in the CAR T-cell therapy commercial landscape underscored the need for repositioning our long-term strategies. In response, SynT expanded its intended application space toward autoimmune diseases, where selectivity and temporal activation are key. This strategic repositioning broadens the impact and relevance of the SynT technology.
Future Outlook
Building on the SynT proof of concept, future work will focus on refining and extending the reporters within collaborative academic networks and, where appropriate, in structured translational programmes. The broader potential of SynT will continue to be explored through the partnerships and consortium efforts established during the project period, and to align our future commercial strategy with the current drastic shift in the cell therapy industry.
Conclusion
SynT has delivered a comprehensive proof of concept for the use of synthetic regulatory DNA elements to monitor CAR T-cell functional states. The project has strengthened computational tools, generated the intended technological assets, and positioned the technology for continued development across oncology and autoimmunity. These achievements advance the broader goal of creating more precise, predictable, and adaptable engineered immune-cell therapies despite the substantial market pressures.
Introducing synthetic regulatory DNA logic to track functional and dysfunctional T-cell states in real time represents a step beyond current cell-therapy engineering standards, establishing a new conceptual basis for designing, assessing, and optimising next-generation immune-cell products.