Chimeric Antigen Receptors (CARs) have shown extraordinary efficacy in numerous clinical trials as an adoptive cell therapy to treat hematological malignancies. Still, CAR T therapy faces significant challenges, ranging from long lead times and expensive manufacturing to complicated vector engineering. Here we describe nano-S/MARt (nS/MARt), a novel DNA vector platform for stable CAR expression with minimal disruption of T cell activity. This antibiotic- free, nanovector technology uses scaffold/matrix attachment regions (S/MARs) for DNA vector maintenance and replication, and transfects primary human T cells efficiently without toxicity. When combined with GMP-compliant MaxCyte Flow Electroporation^® and CliniMACS Prodigy^® automated cell processing, nS/MARt enabled the production of recombinant T cells with stable CAR expression and enhanced anti-tumor activity in only five days. The result was a shortened manufacturing protocol, producing safer cell therapeutics for thousands of patients from a single batch.

Figure 1.  A manufacturing protocol was developed to allow the generation of clinical-grade recombinant T cells using nS/MARt vectors. For this, the CliniMACS Prodigy^® device (Miltenyi^®), a fully automated and closed system for the isolation and culturing of primary human CD3+ cells was coupled with the ExPERT GTx™ large scale electroporation platform (MaxCyte^®).

Figure 2.  A) CD3+ cells were isolated from a leukapheresis product with the TCT process using the CliniMACS Prodigy^®. B) Sorted cells were activated for 3 days with T cell TransActTM, IL-7, and IL-15. On day three 1 x 10 ⁸ cells were counted and electroporated with 125 ug/ml of DNA using the GTx^TM. Shortly after electroporation, the cells were returned to the CliniMACS Prodigy and fed with IL-7- and IL-15 supplemented medium for one day. On day 5, cells were harvested and analyzed by FACS for CAR expression. C) The capability for killing tumor cells was tested in an in vitro killing assay D) and INF-γ production measured.