Periodic Reporting for period 1 - CAR T-REX (CAR T cells Rewired to prevent EXhaustion in the tumour microenvironment)
Okres sprawozdawczy: 2023-04-01 do 2024-06-30
Adoptively transferred T cells expressing recombinant chimeric antigen receptors (CAR T cells) have been approved for the treatment of several haematological malignancies. However, CAR T cell therapy for patients with solid tumours has shown limited benefit so far. One problem is the exhaustion of CAR T cells in the tumour microenvironment (TME). Current state-of-the-art strategies include the permanent modification of CAR T cells to (i) knock out negative regulators (e.g. PD1), or (ii) drive expression of molecules promoting efficacy upon CAR engagement (i.e. TRUCK concept). However, these strategies provide limited adaptation to the location of T cells, and, most importantly, do not limit the activation to the TME where exhaustion should be countered.
In this context, the CAR T-REX project attempts to tackle challenges related to the treatment of solid tumours, aiming at improving efficacy, safety, scalability and cost of therapies.
Genetic rewiring will be achieved through proprietary technology (from TargetGene,TG) to precisely insert artificial miRNAs under endogenous exhaustion-related “Driver” promoters to downregulate “Target” genes that cause exhaustion. These genetic modifications will be implemented using a novel high-performance peptide-based gene delivery platform (developed by Universidade de Santiago de Compostela, USC), allowing combination of several types of cargo. Rewired CAR T cells will be tested on preclinical breast and gastric carcinomas, including humanized animal models (by the Associated Partner University of Debrecen, UD-AP), and variants that eliminate tumours resistant to conventional 2nd and 3rd generation peers (without adverse events) will be developed and manufactured under GMP-like conditions (Stemmatters, STM; and Leibniz Institute for Immunotherapy, LIT), thus accelerating the pathway towards clinical translation.
In this context, the CAR T-REX project attempts to tackle challenges related to the treatment of solid tumours, aiming at improving efficacy, safety, scalability and cost of therapies.
Genetic rewiring will be achieved through proprietary technology (from TargetGene,TG) to precisely insert artificial miRNAs under endogenous exhaustion-related “Driver” promoters to downregulate “Target” genes that cause exhaustion. These genetic modifications will be implemented using a novel high-performance peptide-based gene delivery platform (developed by Universidade de Santiago de Compostela, USC), allowing combination of several types of cargo. Rewired CAR T cells will be tested on preclinical breast and gastric carcinomas, including humanized animal models (by the Associated Partner University of Debrecen, UD-AP), and variants that eliminate tumours resistant to conventional 2nd and 3rd generation peers (without adverse events) will be developed and manufactured under GMP-like conditions (Stemmatters, STM; and Leibniz Institute for Immunotherapy, LIT), thus accelerating the pathway towards clinical translation.
In the first 12 months of the project, the CAR T-REX consortium has made progress within all of the scientific/technical work packages (WP2, WP3, WP4 and WP5) according to the defined work plan.
In WP2 (Genetic modification), the first two (2) milestones have been met, including (i) guide RNA selection, and (ii) design and production of artificial miRNA construct. Additional progress has been made in relation to homology-dependent repair (HDR) in T-cells using the T-GEE platform, and evidence supporting the Driver concept.
The amiRNA polycistron concept has been validated, including knock down of PD1 when driven by endogenous TCRα promoter. A novel miRTron concept has also been proven, with artificial Intron and miRTron properly processed. Expression of the Driver gene was retained as designed and coupled to second protein and amiRNA expression.
In WP3 (Delivery Method Optimisation), libraries of peptides and aldehyde tails with different structural properties have been synthesised. The independent chemical combination of all individual candidates allows for a potential ~ 200 peptide amphiphile carrier library. IVT for optimal mRNA production has been implemented and suitable cell culture and transfection conditions have been optimised for Jurkat cells using Lipofectamine® MessengerMax for benchmarking and a model mRNA cargo (eGFP).
Progress has been made in the screening approach for the USC carrier delivery screening. The full library is now under investigation, with approximately 50% of the screening complete. Preliminary results are being further investigated to maximise efficiency whilst maintaining cell viability. Protocols for the physicochemical characterisation of lead peptide amphiphile carrier molecules and for the formulations with gene material cargos have been developed in line with WP5 requirements.
WP4 (Testing efficacy and safety of rewired CAR T cells) has been focused on optimising assays to evaluate CAR T-cell (i) acute and (ii) expansion/exhaustion-dependent in vitro functions. Work has also been performed on assays involving serial rechallenge until exhaustion (surrogate for high antigen load in solid tumours). The project also includes time-dependent in vivo functions, and for this establishing difficult-to-cure in preclinical models. These models will also be used to investigate epigenomic/transcriptomic changes of regular vs rewired CAR T-cells.
In WP5 (Product compliance and clinical translation), progress has been made on the Quality by Design (QbD) strategy, encompassing the genetic modification (WP2), gene delivery system (WP3) and re-wired CAR T-cells (WP4). A Quality Target Product Profile (QTPP) is under development, covering aspects of general attributes and appearance, safety, purity, identity, quantity, potency.
In WP2 (Genetic modification), the first two (2) milestones have been met, including (i) guide RNA selection, and (ii) design and production of artificial miRNA construct. Additional progress has been made in relation to homology-dependent repair (HDR) in T-cells using the T-GEE platform, and evidence supporting the Driver concept.
The amiRNA polycistron concept has been validated, including knock down of PD1 when driven by endogenous TCRα promoter. A novel miRTron concept has also been proven, with artificial Intron and miRTron properly processed. Expression of the Driver gene was retained as designed and coupled to second protein and amiRNA expression.
In WP3 (Delivery Method Optimisation), libraries of peptides and aldehyde tails with different structural properties have been synthesised. The independent chemical combination of all individual candidates allows for a potential ~ 200 peptide amphiphile carrier library. IVT for optimal mRNA production has been implemented and suitable cell culture and transfection conditions have been optimised for Jurkat cells using Lipofectamine® MessengerMax for benchmarking and a model mRNA cargo (eGFP).
Progress has been made in the screening approach for the USC carrier delivery screening. The full library is now under investigation, with approximately 50% of the screening complete. Preliminary results are being further investigated to maximise efficiency whilst maintaining cell viability. Protocols for the physicochemical characterisation of lead peptide amphiphile carrier molecules and for the formulations with gene material cargos have been developed in line with WP5 requirements.
WP4 (Testing efficacy and safety of rewired CAR T cells) has been focused on optimising assays to evaluate CAR T-cell (i) acute and (ii) expansion/exhaustion-dependent in vitro functions. Work has also been performed on assays involving serial rechallenge until exhaustion (surrogate for high antigen load in solid tumours). The project also includes time-dependent in vivo functions, and for this establishing difficult-to-cure in preclinical models. These models will also be used to investigate epigenomic/transcriptomic changes of regular vs rewired CAR T-cells.
In WP5 (Product compliance and clinical translation), progress has been made on the Quality by Design (QbD) strategy, encompassing the genetic modification (WP2), gene delivery system (WP3) and re-wired CAR T-cells (WP4). A Quality Target Product Profile (QTPP) is under development, covering aspects of general attributes and appearance, safety, purity, identity, quantity, potency.
CAR T-REX aims ultimately at expanding the efficacy, safety and scalability/usability of CAR T technology: (i) efficacy is planned to be addressed by developing novel methods to circumvent CAR T cell exhaustion in the TME; (ii) safety is planned to be addressed through a) the use of a novel, safer gene-editing technique (T-GEE), b) fine-tuning autoregulation of gene-expression in the TME and c) using a novel non-viral delivery system; (iii) scalability and translation readiness is planned to be addressed by incorporating quality-by-design (QbD) methodologies and Good Manufacturing Practices (GMP) principles from project inception.
The consortium has identified key needs to ensure successful development and uptake of the two innovations that are central to the CAR T-REX project, specifically (i) the genetic modification (T-GEE platform), and (ii) delivery method (USC carrier technology, TraffikGene). These needs include:
• Application of technology by end-users;
• End-user feedback;
• Supportive regulatory and standardisation framework, in particular for genetically modified cells;
• Definition of the regulatory roadmap for the implementation of non-viral carriers (excipients) in the CAR T manufacturing process.
The consortium has identified key needs to ensure successful development and uptake of the two innovations that are central to the CAR T-REX project, specifically (i) the genetic modification (T-GEE platform), and (ii) delivery method (USC carrier technology, TraffikGene). These needs include:
• Application of technology by end-users;
• End-user feedback;
• Supportive regulatory and standardisation framework, in particular for genetically modified cells;
• Definition of the regulatory roadmap for the implementation of non-viral carriers (excipients) in the CAR T manufacturing process.