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Autosomally replicating episomal DNA nanovectors allow clinical-scale CAR-T cell manufacturing., The compelling need to provide adoptive cell therapy (ACT) to an increasing number of oncology patients within a meaningful therapeutic window makes the development of an efficient, fast, versatile, and safe genetic tool for creating recombinant T cells indispensable. In this study, we used nonintegrating minimally sized DNA vectors with an enhanced capability of generating genetically modified cells, and we demonstrate that they can be efficiently used to engineer human T lymphocytes. This vector platform contains no viral components and is capable of replicating extrachromosomally in the nucleus of dividing cells, providing persistent transgene expression in human T cells without affecting their behavior and molecular integrity. We use this technology to provide a manufacturing protocol to quickly generate chimeric antigen receptor (CAR)–T cells at clinical scale in a closed system and demonstrate their enhanced anti-tumor activity in vitro and in vivo in comparison to previously described integrating vectors.

The capability to introduce Chimeric Antigen Receptors (CARs) into naïve Human T-Cells represents one of the most promising therapeutic strategies for the treatment of cancer. However, virus mediated adoptive cell therapy (ACT) remain severely limited by two factors: the long lead time and high cost of GMP virus manufacture, and the virus safety profiles. What if the entire ACT process could be sped up, made safer and more cost-effective by at least an order of magnitude? We have invented a novel DNA Vector platform based on scaffold/matrix attachment region (S/MAR) component that provides the opportunity to efficiently generate genetically engineered T-cells. This system is based on a nanovector technology. It contains no immunogenic and comprises only clinically approved sequences. It is easy, simple and cost-efficient to produce. Critically, it does not integrate and replicates autonomously and extrachromosomally in the nuclei of dividing primary human cells, thus avoiding the inherent risk of integrative mutagenesis. Through a process of iterative CpG depletion, selection marker minimalisation, empirical promoter design and elimination of cryptic eukaryotic signals our nano-S/MARt DNA Vector (nS/MARt) can be efficiently transfected into primary human T Cells. nS/MARt vectors are designed to remain stably expressed, and in addition to having the best in class safety profile, they also demonstrate enhanced performance as a biopharmaceutical. Human T-cells engineered to express the CAR receptor against the carcinoembryonic antigen (CEA) using a nS/MARt vector provide more effective killing of human cancer cells in vitro than those engineered with integrative lentivirus. These results hold in vivo, where nS/MARt transfected CAR Tcells outperform the lentivirally transduced cells, attenuating tumour growth and extending mouse survival. Moreover, in pre-clinical studies, the comparison with the FDA approved drug Kymriah®,T cells modified with nS/MARt vectors harbouring the expression of a CD19 CAR are comparable to those engineered with the viral vector. Notably, we have also taken steps to evaluate nS/MARt's scalability and have succeeded in manufacturing a clinically relevant number of CAR-T Cells (2 × 107CAR+ T-cells per kilo, we estimate the production for an individual of 80 Kg). The extension of the results from mice to patients-scale required a 1000x scale up for the processing of T-cell transfection while halving the time for production to hit a meaningful therapeutic window. We have developed a novel manufacturing protocol where nS/MARt vectors can be used "off the shelf" for CAR-T therapy to generate a clinically relevant number of modified cells in just seven days. The delivery of our DNA to CD3+ cells, reaches ~60-70% with cell viability of 60%, that increases in the days that follow the cell electroporation. Thus, the most significant benefit will be for the patients that will be able to access the nS/MARt mediated therapy in 1 week. To translate this technology into a clinical reality a fermentation process that allows the preparation of 2.6 g/L of pure, supercoiled DNA was optimised. There is a pressing need to offer ACT to more oncology patients, and we believe that this novel DNA Vector system provides a unique and innovative approach to this therapeutic strategy for cancer therapy. Citation Format: Matthias Bozza, Alice De Roia, Aileen Berger, Alexandra Tuch, Patrick Schmidt, Richard Harbottle. A non-integrating, non-viral DNA Nanovector platform for the safe, persistent, and rapid manufacture of recombinant T-cells for Adoptive cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4066.

Adoptive immunotherapy is one of the most encouraging therapeutic strategies for the treatment of a range of cancers. One particularly promising avenue of research is the functional introduction of Chimeric Antigen Receptors (CARs) into naive Human T-Cells for autologous-immunotherapy. Currently, the genetic engineering of these cells is achieved through the use of proprietary integrating vector systems such as lentiviruses or the sleeping beauty transposon system which present a risk of genotoxicity associated with their random genomic integration. We have invented a novel DNA Vector platform for the safe and efficient generation of genetically engineered T-Cells for Human Immunotherapy. This DNA vector system contains no viral components and comprises only clinically approved sequences, it does not integrate into the target cell's genome but it can replicate autonomously and extrachromosomally in the nucleus of dividing human primary cells. These DNA Vectors offer several advantages over currently used vector systems; they are not subject to commercial licences, they are cheaper and easier to produce, and they can more quickly genetically modify human cells without the inherent risk of integrative mutagenesis. In preclinical experiments we have successfully generated genetically engineered human T-Cells expressing the CAR receptor against several epitopes and have demonstrated their viability and capability in targeting and killing human cancer cells which express these epitopes. The long term anti-tumor activity of our DNA-CAR-T cells has been confirmed in vivo using xenotransplanted cell lines in immunodeficient mice. We believe that this novel DNA Vector system provides a unique and innovative approach to this exciting therapeutic strategy for cancer therapy. We estimate that this novel methodology will provide a simpler method of CAR T-cell manufacturing, resulting in a 10-fold reduction in the cost of the CART-product. Citation Format: Patrick Schmidt, Matthias Bozza, Aileen Berger, Claudia Luckner-Minden, Alexandra Tuch, Inka Zörnig, Dirk Jäger, Richard Harbottle. Novel DNA vectors encoding a chimeric antigen receptor mediate long term expression without genomic integration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3573.