Identification of small molecules that enhance transduction efficiency in human T cells
Louise Treanor1, Aaron Wilson2, Yuan Wang2, Amy Shaw, Nathan Ross2, John Tallarico2, Mark Dudley and Jennifer Brogdon1
1Department of Immuno-oncology and the 2Department of Molecular Pathways, Novartis Institute for Biomedical Research, Cambridge, MA 02139
Transduction of human T cells is an integral component of Chimeric antigen T cell receptor (CART) therapy. The introduction of the gene of interest via lentiviral infection is essential to the generation of the cell product, thus optimization of this stage is critical to the overall success of the therapy. Thus we designed and executed a high throughput screen that optimized the transduction of primary human T cells using a library of annotated compounds that constitutes a library of over 3000 compounds with known targets or mechanisms of action.
A scaled down assay of the nine day WAVE bioreactor CART process was developed that was acutely sensitive to perturbations in transduction efficiency. It encompassed culturing primary human T cells over 4 days in 384 well plates with an optimized T cell density, bead to T cell ratio and the multiplicity of infection of the virus. The output of the primary screen was viability, CD4/CD8 ratio and T cell phenotype of a single human T cell donor screened in duplicate in 3 different concentrations.
Approximately 70 compounds that increased transduction at least 2 fold in a multiple concentrations were identified as hits from the primary screen. A collection of 200 compounds were assembled for an eight point dose response assay that either targeted the same pathway or were structurally similar to the 70 parent compounds. Within this collection, targeting three specific pathways in two independent human T cell donors emerged as the most effective means to improve transduction efficiency. More compounds, including some that are in late stage clinical development, that target these specific pathways were screened in a 96 well plate assay over nine days. Two pathways emerged as the most effective targets and at least a 3 fold enhancement of transduction efficiency was observed in 4 independent human T cell donors.