Adoptive immunotherapy is one of the most encouraging therapeutic strategies for the treatment of a range of cancers. A 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 integrating vector systems such as lentiviruses or the sleeping beauty transposon system, which present a potential 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 nuclei of dividing human primary cells. These DNA vectors offer several advantages over currently used vector systems; they are not subject to commercial licenses, 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 that sustain the expression of a reporter gene for over a month at persistently high levels without decline. We have also successfully modified these cells with a range of transgenic CAR receptors against several known cancer cell epitopes and we have demonstrated their viability and capability in the targeted killing of these human cancer cells. We showed that CAR-T-cells generated with our technology killed more efficiently target cells when compared to T-cells engineered with current state-of-the-art integrative lentivirus. The expression of functional CARs was detected over a period of two weeks of administration in culture and the anticancer activity of our DNA-CAR-T-cells was evaluated in vivo using xenotransplanted cell lines in immunodeficient mice. We are currently performing analyses in order to determine the molecular behavior of the vector in the cells and its impact on cellular viability. Furthermore, we are developing a protocol for large scale electroporation in order to manufacture a clinical grade CAR-T DNA product. 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 CAR-T product.
Citation Format: Patrick Schmidt, Matthias Bozza, Dirk Jaeger, Richard Harbottle. A novel nonviral, nonintegrative DNA vector system for T-cell engineering [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A042.