Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CARAT (Chimeric Antigen Receptors (CARs) for Advanced Therapies)

Teaser

Overall, the CARAT (“Chimeric Antigen Receptors for Advanced Therapies”) project is aiming at a particular breakthrough in cellular cancer therapy through the delivery of a comprehensive platform enabling automated, easy-to-use and cost-efficient manufacture of effective...

Summary

Overall, the CARAT (“Chimeric Antigen Receptors for Advanced Therapies”) project is aiming at a particular breakthrough in cellular cancer therapy through the delivery of a comprehensive platform enabling automated, easy-to-use and cost-efficient manufacture of effective CAR-modified T cells for personalized cell therapy as outlined in Figure 1. Specifically, the project aims to develop advanced enabling technologies. CAR therapy needs more than cutting-edge tools to achieve a clinical breakthrough. In this respect, more effective T cells are being designed next to innovative next-generation CARs that enable better control and higher safety. Concomitantly, gene-delivery of CARs into T cells is being optimized. Novel monitoring technologies are 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.

Work performed

Partner Ospedale San Raffaele (OSR) optimized the culture conditions for the generation of biologically active CAR-T cells: They defined the right timing for T cell transduction, investigated the phenotypic and functional changes encountered when starting from different cell populations, implemented their Institutional Biobank with additional blood samples from ALL patients and, finally, generated an EGFR CAR that will be used as a model CAR for targeting solid tumours. Miltenyi optimized T cell isolation and demonstrated that the way of enriching T cells has an impact on the transduction efficiency with lentiviral vectors. Moreover, harmonization efforts of T cell expansion conditions for the manual and automated process have taken place in order to generate early differentiated T cells (namely Tcm and Tscm).
A panel of 2nd generation combination and split signal CAR constructs were designed and generated by molecular cloning at University College London (UCL). Clear evidence confirming the proof-of-principle was achieved demonstrating successful drug-mediated CAR inhibition with clear CAR-mediated cytotoxicity apparent in the absence of the drug. 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. CAR sharing was established via a 1st standard CAR (for liquid malignancies) by Miltenyi and will be used amongst partners who already possess a range of CARs to be compared and even optimized in CARAT towards better efficacy.
Partner Paul-Ehrlich-Institute (PEI) could generate a panel of lentiviral vectors (LV) that mediate selective gene transfer into T cell subtypes and could achieve targeting of resting T cells. University Hospital Freiburg (UKL-FR) generated designer nucleases that target the TCR encoding loci and established functional assays to evaluate T cell activity. Towards the generation of AAV vectors that deliver checkpoint inhibitors exclusively to tumour tissue, AAV transfer vectors encoding mini antibodies recognizing mouse PD-1 or PD-L1 were generated and tested.
First in vitro imaging experiments were performed at Cochin Institute (INSERM) looking at the interactions between RAJI B cells and CD20 CAR T cells. A potent in vitro activity of CD20 CAR T cells could be demonstrated. The ability of CAR T cells to stably interact with tumour cells and increase their Ca2+ could also be shown. Moreover, using cryo-sliced material as specimen for multi-staining purposes (MICS) at Miltenyi feasibility and proof of principle results could be obtained.
Miltenyi Biotec has released to market a first version of application software (=program controlling the automated manufacturing) enabling the automated manufacturing of lentivirally modified T cells on a single platform. Miltenyi has confirmed that CAR T cells generated on the Prodigy platform are functional in vivo using currently optimized conditions. The platform is increasingly adopted in the field for clinical use. Moreover, 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. Partner TrakCel, working with Miltenyi, 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 platforms (e.g. Prodigy, MACSquant analyser and TrakCel platform) have been defined and the required programming work is ongoing.
Partner Eurice coordinated the setup of the project website (www.carat-horizon2020.eu). Initial activities relating to an increased visibility include a set of press releases announcing the launch of the project and various other articles in the media. The CARAT consortium partners have started to raise awareness of the project’s mission and vision within the scientific community by giving a first insight into the project at international conferences. In p

Final results

CARAT partners perform their work with a clear focus on technological innovation, translation and exploitation. In this regard, the consortium has set 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. This key innovation of CARAT will be of worldwide relevance because it will reliably perform in an automated manner, with minimal user interaction, all the manufacturing steps in an entirely closed system and therefore enable the field to further develop cell therapies for clinical use.
• To demonstrate proof-of-concept and regulatory compliance. The CARAT platform will be designed for full GMP compliance to fulfil regulatory requirements at a thus far unreached level. Automation will improve reproducibility and standardization as well as transferability; integrated in-process control and innovative tracking logistics will result in a significantly reduced risk of handling errors.
• To disseminate broadly applicable, simplified CAR T cell technologies. We aim to deliver CAR therapies not only to the pharmaceutical industry or to specialized university clinics but also to enable localized “cottage” hospitals to perform innovative personalized cancer therapies based on the CARAT platform.

Website & more info

More info: http://carat-horizon2020.eu/.