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.

We describe herein non-integrating minimally sized nano-S/ MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patientderived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.

Within the last decade CAR-T cells have changed the landscape of treatment regimen for leukemia and myeloma, which is reflected by the first FDA approval of this class of living drugs in 2017. As of today, the clinical trial situation aims on CAR-T applicability in solid tumors, which is more arduous due to antigen heterogeneity and limited CAR-T persistence. More personalized and multi-target oriented cellular products may offer a solution to overcome these problems but then CAR identification and selection display the major bottleneck in the drug development process. Usually, matching scFvs are selected from targeting screens of a phage-display library and hits are subsequently cloned in CAR backbones and tested for functionality and possible limiting factors as the occurrence of tonic signaling. This makes the whole process very time consuming and laborious. Within this project, we propose a novel CAR-T selection method that rapidly shortens the discovery procedure. We have developed a full length CAR library in nS/MARt DNA vectors that is electroporated in a Jurkat reporter cell line reflecting entirely its full diversity. By this, we can quickly identify the amount of tonic signaling CARs and exclude them from further selection. For on-target selection we first perform a bulk pre-selection followed by a single cell functionality screening using the Berkeley Lights Lightning™ device. This allows us to export hits as clonal viable cells that undergo long length Nanopore CAR-RNA sequencing. Our findings show the feasibility of our approach and that it can shorten the timeframe needed for the full selection process from weeks to days. Citation Format: Eren Boga, Luisa Berger, Alice De Roia, Inka Zörnig, Robert Embacher, Stefan B. Eichmüller, Dirk Jäger, Richard Harbottle, Patrick Schmidt. A combination approach of a cellular library and single cell microfluidics analysis for the rapid selection of CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1769.