Periodic Reporting for period 3 - CARAT (Chimeric Antigen Receptors (CARs) for Advanced Therapies)
Reporting period: 2019-01-01 to 2019-12-31
The CARAT (“Chimeric Antigen Receptors for Advanced Therapies”) project aimed at a particular breakthrough in cellular cancer therapy through the delivery of a comprehensive platform enabling automated, easy-to-use and cost-effective manufacture of potent CAR-modified T cells for personalized cell therapy (Figure 1). Specifically, the project aimed to develop advanced enabling technologies. CAR therapy needs more than cutting-edge tools to achieve a clinical breakthrough. In this respect, more efficient T cells have been designed next to innovative next-generation CARs that enable better control and higher safety. Concomitantly, gene-delivery of CARs into T cells has been optimized. Novel monitoring technologies were applied to understand the mode of action of CAR T cells, and specific logistic technologies are allowing for better control of the entire chain of custody during the manufacturing process.
Partner Ospedale San Raffaele (OSR) worked to improve the antitumor efficacy of CAR T cell therapy by ameliorating T cell fitness. T cell expansion and persistence after perfusion can be enhanced by expanding early memory CAR T cells. This small scale finding was translated to an automated clinical procedure. Partner OSR found out that applying the optimized procedure to specific T cell subpopulations, can further improve the system. Strikingly, they observed in animal experiments that CAR T cells generated from selected T cell subsets have superior antitumor activity, being able to mediate profound primary responses and to protect mice from disease recurrence. They showed that these cells have an intrinsically lower potential to cause detrimental infusion toxicities. A panel of 2nd generation combination and split signal CAR constructs were designed and generated by molecular cloning at University College London (UCL) in order to enable improved control of CAR T cell function or persistence. Clear evidence of drug-mediated CAR inhibition proof-of-principle was achieved. Two independent means to control CAR T cell function by either disrupting the signalling of the CAR through a clinically well known drug (allowing for a more tightly controlled inflammatory response driven by the CAR T cells) as well as by eliminating the CAR T cells when they are no longer required. Further, UCL designed and generated proof-of-principle signal hijack by molecular cloning in order to enhance CAR efficacy in the presence of a PD1 expressing tumor.
Partner Paul-Ehrlich-Institute (PEI) has achieved the generation of CAR T cells under minimally activating conditions. Instrumental for this success was the surface engineering of lentiviral vectors resulting in the use of cell surface proteins present on minimally activated T lymphocytes for cell entry. (). This is an important progress in CAR T cell manufacturing, since it has the potential to simplify the currently very complex process by reducing processing steps and overall production time.
Partner PEI generated and assessed a novel tumor-targeted vector based on adenovirus-associated virus (AAV) as vehicle for delivery of checkpoint inhibitors. ). By this means, tumor-targeted AAVs can redirect the biodistribution of checkpoint inhibitors from liver and serum to tumor tissue and can be combined with other types of immunotherapy including CAR T cells. This strategy offers the potential to make this therapy safer for cancer patients Universitätsklinikum Freiburg (UKL-FR)’s aim was to improve efficacy and safety of CAR T cells as well as to develop novel CARs to target a solid tumor. To prevent alloreactivity and to minimize off-tumor effects of CAR T cells, an efficient gene editing approach was designed to disrupt a locus encoding the T cell receptor (TCR) in an automated GMP-compliant manufacturing process to produce TCR-negative CAR T cells. To improve CAR T cell activity in the hostile microenvironment of a solid tumor, several gene editing approaches to disrupt loci encoding immune checkpoint inhibitors, such as PD-1, were developed. UKL-FR generated novel CARs to target the prostate-specific membrane antigen and showed their effect in animal models (PSMA). Animal experiments showed, that Cochin Institute (INSERM) has established a preclinical model named “tumor slice assay” which enables the monitoring of CAR T cells . INSERM has implemented an organotypic model based on thick slices (0.5 mm) of human tumors (lung and renal) kept fresh during the entire experiment. This approach, combined with dynamic imaging microscopy, made it possible to track CAR T cells in a preserved human tumor environment. This approach uncovered a new mechanism by which CAR T cells infiltrate islets of carcinomas in a two-step process with a key role of IFNg and ICAM-1.. This method is considered to be a valuable model to predict CAR T cell efficacy but also safety and the influence of tumor microenvironment on CAR T cells.
Miltenyi Biotec has released to market a first version of an application enabling the automated manufacturing of lentivirally modified T cells on a single closed-platform. Miltenyi has defined panels of markers for automated flow analysis of the CAR T cells in order to define cell composition, CAR transduction efficiency and viability. Successful clinical validation of the process in multicentric clinical trials contributed to easy and affordable access to CAR T cell therapies by bringing the cell products closer to the point of care and reducing logistical complexity.
In order to give guidance to the scientific community towards clinical manufacturing submissions multiple supportive documents were developed. Partner TrakCel, working with Miltenyi Biotec, has made great progress by building process maps for a generic logistic template for a CAR-T therapy. Solutions for the exchange of information between manufacturers, transport teams and treating centres were developed and tested at customer’s site. The feedback received after the training has contributed to making TrakCels’s software more user friendly and has improve its implementation. Partner Eurice has constantly updated the project website (www.carat-horizon2020.eu) with news from the project and the larger scientific community, including announcements of relevant conferences. An animated video was produced to introduce CARAT to different audiences and published on the projects website. CARAT dissemination material was produced and displayed at various occasions.
Partner Paul-Ehrlich-Institute (PEI) has achieved the generation of CAR T cells under minimally activating conditions. Instrumental for this success was the surface engineering of lentiviral vectors resulting in the use of cell surface proteins present on minimally activated T lymphocytes for cell entry. (). This is an important progress in CAR T cell manufacturing, since it has the potential to simplify the currently very complex process by reducing processing steps and overall production time.
Partner PEI generated and assessed a novel tumor-targeted vector based on adenovirus-associated virus (AAV) as vehicle for delivery of checkpoint inhibitors. ). By this means, tumor-targeted AAVs can redirect the biodistribution of checkpoint inhibitors from liver and serum to tumor tissue and can be combined with other types of immunotherapy including CAR T cells. This strategy offers the potential to make this therapy safer for cancer patients Universitätsklinikum Freiburg (UKL-FR)’s aim was to improve efficacy and safety of CAR T cells as well as to develop novel CARs to target a solid tumor. To prevent alloreactivity and to minimize off-tumor effects of CAR T cells, an efficient gene editing approach was designed to disrupt a locus encoding the T cell receptor (TCR) in an automated GMP-compliant manufacturing process to produce TCR-negative CAR T cells. To improve CAR T cell activity in the hostile microenvironment of a solid tumor, several gene editing approaches to disrupt loci encoding immune checkpoint inhibitors, such as PD-1, were developed. UKL-FR generated novel CARs to target the prostate-specific membrane antigen and showed their effect in animal models (PSMA). Animal experiments showed, that Cochin Institute (INSERM) has established a preclinical model named “tumor slice assay” which enables the monitoring of CAR T cells . INSERM has implemented an organotypic model based on thick slices (0.5 mm) of human tumors (lung and renal) kept fresh during the entire experiment. This approach, combined with dynamic imaging microscopy, made it possible to track CAR T cells in a preserved human tumor environment. This approach uncovered a new mechanism by which CAR T cells infiltrate islets of carcinomas in a two-step process with a key role of IFNg and ICAM-1.. This method is considered to be a valuable model to predict CAR T cell efficacy but also safety and the influence of tumor microenvironment on CAR T cells.
Miltenyi Biotec has released to market a first version of an application enabling the automated manufacturing of lentivirally modified T cells on a single closed-platform. Miltenyi has defined panels of markers for automated flow analysis of the CAR T cells in order to define cell composition, CAR transduction efficiency and viability. Successful clinical validation of the process in multicentric clinical trials contributed to easy and affordable access to CAR T cell therapies by bringing the cell products closer to the point of care and reducing logistical complexity.
In order to give guidance to the scientific community towards clinical manufacturing submissions multiple supportive documents were developed. Partner TrakCel, working with Miltenyi Biotec, has made great progress by building process maps for a generic logistic template for a CAR-T therapy. Solutions for the exchange of information between manufacturers, transport teams and treating centres were developed and tested at customer’s site. The feedback received after the training has contributed to making TrakCels’s software more user friendly and has improve its implementation. Partner Eurice has constantly updated the project website (www.carat-horizon2020.eu) with news from the project and the larger scientific community, including announcements of relevant conferences. An animated video was produced to introduce CARAT to different audiences and published on the projects website. CARAT dissemination material was produced and displayed at various occasions.
CARAT partners perform their work with a clear focus on technological innovation, translation and exploitation. In this regard, the consortium has reached its goals as follows:
•To assemble tools and technologies towards an integrated CARAT process for automated GMP-compliant manufacture of more effective T cells for CAR therapy. As a main impact of CARAT, the platform has set standards in the industry for robust and reproducible automated manufacturing of CAR T cells
and therefore enable the field to further bring cell therapies to the clinic.
•To demonstrate proof-of-concept and regulatory compliance. The CARAT platform is designed for full GMP compliance to fulfil regulatory requirements at a thus far unreached level.
•To disseminate broadly applicable, simplified CAR T cell technologies. This will greatly increase the dynamics around cell therapies in Europe and promote scientific innovation and industrial developments as a whole and potentially attract global actors to Europe.
•To assemble tools and technologies towards an integrated CARAT process for automated GMP-compliant manufacture of more effective T cells for CAR therapy. As a main impact of CARAT, the platform has set standards in the industry for robust and reproducible automated manufacturing of CAR T cells
and therefore enable the field to further bring cell therapies to the clinic.
•To demonstrate proof-of-concept and regulatory compliance. The CARAT platform is designed for full GMP compliance to fulfil regulatory requirements at a thus far unreached level.
•To disseminate broadly applicable, simplified CAR T cell technologies. This will greatly increase the dynamics around cell therapies in Europe and promote scientific innovation and industrial developments as a whole and potentially attract global actors to Europe.