Periodic Reporting for period 1 - ArTCell (Bottom-up manufacturing of artificial anti-tumor T cells)
Período documentado: 2024-02-01 hasta 2025-01-31
The ArTCell project aims to drive a paradigm shift in cancer immunotherapy by developing a scalable, on-demand artificial T-cell substitute with cytotoxic functionalities. This novel approach seeks to engineer an advanced synthetic cell capable of executing T-cell-mediated tumor eradication while circumventing the limitations associated with conventional cell-based therapies. By leveraging controlled raw materials, this platform enables the mass production of a safe, off-the-shelf, and cost-effective therapeutic solution, thereby addressing the cost, manufacturing complexity, and donor-dependent limitations of current allogeneic CAR-T cell therapies.
At the core of this technology lies the construction of giant unilamellar vesicle (GUV) that recapitulates the biophysical and mechanical properties of a natural cell. This engineered vesicle will be equipped with a tumor-recognizing chimeric antigen receptor (CAR) and the essential effector molecules required for cytotoxic function. By entirely bypassing the use of cell-derived components, ArTCell introduces a disruptive innovation in immunotherapy, offering a solution to major challenges such as immune rejection and the inherent variability of biological cell sources.
In the initial phase of the project, we established the foundational components necessary for artificial T-cell construction. Specifically, we focused on isolating and integrating critical membrane proteins (MPs) required for T-cell effector function while simultaneously optimizing the GUV cytoskeletal framework. This structural reinforcement ensures mechanical stability and functionality, forming the basis for a fully operational synthetic immune cell. Our work over the first year has laid the groundwork for further development, ultimately advancing toward a clinically viable immunotherapeutic platform within the project's five-year timeline.
At the core of this technology lies the construction of giant unilamellar vesicle (GUV) that recapitulates the biophysical and mechanical properties of a natural cell. This engineered vesicle will be equipped with a tumor-recognizing chimeric antigen receptor (CAR) and the essential effector molecules required for cytotoxic function. By entirely bypassing the use of cell-derived components, ArTCell introduces a disruptive innovation in immunotherapy, offering a solution to major challenges such as immune rejection and the inherent variability of biological cell sources.
In the initial phase of the project, we established the foundational components necessary for artificial T-cell construction. Specifically, we focused on isolating and integrating critical membrane proteins (MPs) required for T-cell effector function while simultaneously optimizing the GUV cytoskeletal framework. This structural reinforcement ensures mechanical stability and functionality, forming the basis for a fully operational synthetic immune cell. Our work over the first year has laid the groundwork for further development, ultimately advancing toward a clinically viable immunotherapeutic platform within the project's five-year timeline.
SO 1: Purification of native & recombinant T cell proteins
During the first year of ArTCell project, P3 successfully started developing recombinant proteins relevant to the T cell system, incorporation of which into the structure of GUVs will allow us to recapitulate three different ArTCell models endowed with the T cell-like functionality to target B cell-derived and myeloid blood tumors. The objective of WP1 is hence purification of recombinant functional T cell proteins. Achieving this objective includes working on three specific Tasks: Task 1.1 (relevant to D1.1): Expression and isolation of recombinant membrane proteins (MPs) Task 1.2 (relevant to D1.2): Expression and purification of recombinant pore-forming proteins Task 1.3 (relevant to D1.3): Evaluation biological activity of isolated proteins
Activities relevant to these tasks have been split into two separate sub-packages corresponding to the two major ARTCELL prototypes envisioned in the working plan, specifically
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the model A/B functioning via adhesion-dependent killing and including recombinant integrin LFA-1, cytotoxic death ligand TRAIL, and CARs specific either for the B cell marker CD19 (model A) or the myeloid/myeloma marker CD38 (model B). Working on this model includes activities of the Task 1.1 and Task 1.3.
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the model C functioning via adhesion-release-dependent killing and including recombinant CARs intracellularly coupled to a triggerable synthetic signaling module composed of caspase-1 p46 (termed hereafter “CAR-p46”) and two cytotoxic pore-forming proteins. Working on this model includes activities of all three Tasks 1.1-1.3.
The work on model A/B proteins has proceeded smoothly. We fine-tuned each separate recombinant construct to ensure its functional properties and expression levels on the surface of
CHO cell lines. This was achieved for all constructs involved (CD19 CAR, CD38 CAR, LFA-1, TRAIL). Additionally, we set up experimental conditions for evaluation of biologic activity of isolated proteins, such as the promotion of binding to target cells and cytotoxic killing. Stable cell lines have been already generated for CD19 CAR receptor and transferred to P1 to initiate MP purification and GUV production. Generation of stable cell lines for remaining constructs is expected to be complete by Month 15 of the project (in line with D1.1). Although we could isolate CAR CD19 within nanodiscs (NDs, but purification with HisTrap affinity column by AKTA system was not promising. So, we are optimising protocol for purification the CAR CD19-enriched NDs using StrepTrap column. Hence, work on model A/B proteins is proceeding according to the established plan.
The work on model C proteins has been undertaken in parallel. Development of recombinant constructs for this ArTCell prototype necessitates refinement because the initial design of the CAR-p46 protein was characterized by high cytotoxicity. This shortcoming (anticipated as Critical Risk #2 in the working plan) is being addressed through two alternative strategies.
SO2: Improving mechanical stability and reducing immunogenicity
Partner 4 has made progress in developing various hydrogel-based strategies to engineer a supporting structure with well-defined mechanical properties for GUV stabilization. To achieve this, the production of single and double emulsion droplets has been successfully demonstrated using co-flow, co-flow, and co-flow focusing microfluidic devices. The fabrication of these devices, along with the challenges encountered during the process, has been carefully documented. Among the configurations, the co-flow focusing setup shows promising potential for GUV formation, offering controlled droplet generation under optimized conditions.For the incorporation of hydrogels into GUVs, the formation of ionically crosslinked droplets composed of sodium alginate (SA) and polysaccharides using ZnCl2 and FeCl₃ was explored but proved suboptimal in terms of efficiency and droplet uniformity. As an alternative, the use of UV-mediated gelatin methacrylate was investigated, yielding spherical, monodisperse droplets without encountering clogging issues. This approach has shown promising preliminary results, indicating the potential for more controlled and reproducible droplet formation..
During the first year of ArTCell project, P3 successfully started developing recombinant proteins relevant to the T cell system, incorporation of which into the structure of GUVs will allow us to recapitulate three different ArTCell models endowed with the T cell-like functionality to target B cell-derived and myeloid blood tumors. The objective of WP1 is hence purification of recombinant functional T cell proteins. Achieving this objective includes working on three specific Tasks: Task 1.1 (relevant to D1.1): Expression and isolation of recombinant membrane proteins (MPs) Task 1.2 (relevant to D1.2): Expression and purification of recombinant pore-forming proteins Task 1.3 (relevant to D1.3): Evaluation biological activity of isolated proteins
Activities relevant to these tasks have been split into two separate sub-packages corresponding to the two major ARTCELL prototypes envisioned in the working plan, specifically
•
the model A/B functioning via adhesion-dependent killing and including recombinant integrin LFA-1, cytotoxic death ligand TRAIL, and CARs specific either for the B cell marker CD19 (model A) or the myeloid/myeloma marker CD38 (model B). Working on this model includes activities of the Task 1.1 and Task 1.3.
•
the model C functioning via adhesion-release-dependent killing and including recombinant CARs intracellularly coupled to a triggerable synthetic signaling module composed of caspase-1 p46 (termed hereafter “CAR-p46”) and two cytotoxic pore-forming proteins. Working on this model includes activities of all three Tasks 1.1-1.3.
The work on model A/B proteins has proceeded smoothly. We fine-tuned each separate recombinant construct to ensure its functional properties and expression levels on the surface of
CHO cell lines. This was achieved for all constructs involved (CD19 CAR, CD38 CAR, LFA-1, TRAIL). Additionally, we set up experimental conditions for evaluation of biologic activity of isolated proteins, such as the promotion of binding to target cells and cytotoxic killing. Stable cell lines have been already generated for CD19 CAR receptor and transferred to P1 to initiate MP purification and GUV production. Generation of stable cell lines for remaining constructs is expected to be complete by Month 15 of the project (in line with D1.1). Although we could isolate CAR CD19 within nanodiscs (NDs, but purification with HisTrap affinity column by AKTA system was not promising. So, we are optimising protocol for purification the CAR CD19-enriched NDs using StrepTrap column. Hence, work on model A/B proteins is proceeding according to the established plan.
The work on model C proteins has been undertaken in parallel. Development of recombinant constructs for this ArTCell prototype necessitates refinement because the initial design of the CAR-p46 protein was characterized by high cytotoxicity. This shortcoming (anticipated as Critical Risk #2 in the working plan) is being addressed through two alternative strategies.
SO2: Improving mechanical stability and reducing immunogenicity
Partner 4 has made progress in developing various hydrogel-based strategies to engineer a supporting structure with well-defined mechanical properties for GUV stabilization. To achieve this, the production of single and double emulsion droplets has been successfully demonstrated using co-flow, co-flow, and co-flow focusing microfluidic devices. The fabrication of these devices, along with the challenges encountered during the process, has been carefully documented. Among the configurations, the co-flow focusing setup shows promising potential for GUV formation, offering controlled droplet generation under optimized conditions.For the incorporation of hydrogels into GUVs, the formation of ionically crosslinked droplets composed of sodium alginate (SA) and polysaccharides using ZnCl2 and FeCl₃ was explored but proved suboptimal in terms of efficiency and droplet uniformity. As an alternative, the use of UV-mediated gelatin methacrylate was investigated, yielding spherical, monodisperse droplets without encountering clogging issues. This approach has shown promising preliminary results, indicating the potential for more controlled and reproducible droplet formation..