Electroporation of a Non-Integrative DNA Nanovector for Efficient, Semi-Automated, GMP Manufacturing of CAR T Cell Therapies
Recombinant T cell expression of Chimeric Antigen Receptors (CARs) has 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. Also, CAR T cell production routinely employs random integration of viruses or transposons, which carries an inherent risk of genotoxicity and costly, long-term patient follow-up. CAR engineering by transient mRNA transfection could be safer but more cost-prohibitive, requiring several doses per patient. DNA is a promising alternative but can cause sensitive T cells to lose functional capacity or induce apoptosis.
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 and 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.
Optimized Nanovectors Provide Prolonged Transgene Expression in Primary CD3+ Cells
nS/MARt Delivery by Electroporation Has Minimal Impact on Human T Cells and Provides Superior Functionality
nS/MARt T Cells Mediate Efficient Tumor Killing in vivo
Development of GMP-Compatible, Large-Scale CAR T Cell Manufacturing with nS/MARt Vectors
Enabling function of ExPERT GTx Flow Electroporation:
- Efficient and consistent cargo delivery with more than 80% of viable cells expressing reporter gene.
- Electroporation did not alter CD4:CD8 ratio (compared to mock electroporated cells).
- Electroporation did not impair T cell proliferation (compared to unpulsed CD3+ cells).
- Minimal disruption of transcriptomic profile, electroporated cells displayed a more naïve phenotype.
- More active and highly efficient killing capabilities of T cells.
- Large scale MaxCyte Flow Electroporation allowed to modify 3 x 108 cells yield 50-75% viability and 65-80% efficiency (donor dependent).
- MaxCyte electroporation proved to be gentle, and when combined with a highly optimized vector, resulted in cells that were more potent at eliminating tumors than standard LV-based therapies.