Third-Generation Human Epidermal Growth Factor Receptor 2 Chimeric Antigen Receptor Expression on Human T Cells Improves with Two-Signal Activation.

Patient derived T cells activated ex vivo with CD3/CD28 beads show superior expansion. Therefore, CD3/CD28 beads have huge potential to be used in the clinic for immunotherapy applications. Two protocols were devised to evaluate if the expression of third-generation human epidermal growth factor receptor 2 chimeric antigen receptor (CAR) can be improved on human T cells activated with CD3/CD28 beads. In protocol 1, unconcentrated human epidermal growth factor receptor 2 CAR retroviral supernatants were used, and in protocol 2, concentrated virus was used. The results demonstrate that compared to unconcentrated viral supernatants, transduction with the concentrated virus improved the infection rate of bead activated CD4 T cells from ∼40% to ∼70%, and the fluorescent intensity values improved from ∼12,000 to ∼28,000 mean fluorescence intensity units. These results demonstrate the utility of these protocols for CAR immunotherapies.