705. Enhancement of CAR Expression of piggyBac Transposon-Engineered T Cells by Stimulation with Viral Antigens
Authors: Daisuke Morita, Miyuki Tanaka, Nobuhiro Nishio, Nozomu Kawashima, Yusuke Okuno, Matthew H. Wilson, Cliona M. Rooney, Yoshiyuki Takahashi, Yozo Nakazawa
Journal: Molecular Therapy (2016)

Introduction We previously showed that the piggyBac transposon system is a promising genetic tool for stable, non-viral gene engineering of primary human T cells. Our initial culture strategy for expanding piggyBac-modified CD19 CAR-T cells required magnetic cell isolation due to low CAR expression and 21 days of culture and our CAR included an IgG1-Fc spacer that may limit CAR-T cell expansion in vivo. We have now improved these process and construct to avoid magnetic cell isolation, shorten the culture period, and increase in vivo efficacy. Methods We used a 4D-Nucleofector device to electroporate 1 × 107 peripheral blood mononuclear cells from 9 healthy donors with a CD19.CD28.ζ–CAR transposon plasmid, with or without an IgG1-CH2CH3 spacer and a piggyBac transposase plasmid. We compared 2 new culture conditions with our original method for expansion of CD19 CAR-T cells. All cells were cultured in serum-free medium (TexMACS) containing IL-7 and IL-15 in 24-well plates. Electroporated cells were immediately transferred to irradiated autologous activated T-cells (ATCs), either pulsed with 4 viral peptide pools (PepTivator; AdV5 Hexon, CMV pp65, EBV EBNA-1, and BZLF1) (ACE), or unpulsed (non-ACE). The next day, non-ACE cells were transferred to CD3 and CD28 antibody-coated plates for 5 days. In the original method, electroporated cells were transferred to CD3/CD28-coated plates one day after electroporation without ATCs. On day 7, all cells were transferred into G-Rex10 culture flasks with ACE-pulsed or unpulsed irradiated ATCs or no ATCs for ACE, non-ACE and original cells respectively. On day 14, we collected CAR-T cells from all conditions. Results We obtained 4.7 ± 3.0 × 107 ACE CAR-T cells, 6.7 ± 2.1 × 107 non-ACE CAR T cells, and 5.3 ± 2.3 × 107 original CAR T cells after 14 days of culture. ACE CAR-T cells showed significantly higher expression of the CAR transgene (33.0 ± 9.7% and >50% for CD19.CAR with and without the CH2CH3 spacer respectively) than non-ACE (10.7 ± 7.8%) or original CAR-T cells (4.6 ± 3.2%). All CAR-T-cells inhibited the growth of CD19+ tumors (95%, 60% and 51% for ACE, non-ACE and original CAR-T at an E:T ratio of 1:50; ACE CAR-T cells contained 29.6 ± 17.3% CD4+ cells and 64.3 ± 16.8% CD8+ cells, with 79.9 ± 5.9% cells co-expressing CD45RA and CCR7 and 62.6 ± 20.9% cells co-expressing CD45RO and CD62L. In an in vivo mouse model, ACE CAR-T cells delayed tumor growth by 30 days compared to no T-cells. Conclusions We markedly increased CAR expression from piggyBac-mediated CD19 CAR-T cells without cell selection, reducing the culture period to 14 days using viral antigen stimulation. Deletion of IgG1-Fc spacer led to further increase of CAR expression. Together with the simple manufacturing process and cost-effectiveness of DNA transposon technology, piggyBac-based gene transfer provides an alternative to retro- or lentiviral gene transfer for CAR T-cell therapy.