Background Chimeric antigen receptor (CAR) T-cells are engineered immune cells that can be taken from a patient and redirected to fight cancer. Recently, we developed a virus-free process to create anti-GD2 CAR T cells using CRISPR-Cas9 ribonucleoproteins that endows cells with a stem cell memory-like and less exhausted phenotype that improves their ability to induce regression of GD2 positive solid tumors.

Methods To improve this workflow, we systematically modified the media conditions, electroporation timing, and small molecule treatment to improve the cell yield and memory-like characteristics of CAR T cells.

Results We find that optimal non-viral knock-in is dependent on activation time, and two key small molecule inhibitors.

Conclusions Key reagents have been made GMP-compatible, and therefore this process could be readily implemented in existing cell manufacturing facilities.

Ethics Approval The study was approved by the IRB at University of Wisconsin-Madison, approval number 2018-0103