Table 3.

Manufacturing protocols associated with preclinical studies employing PB in the context of CAR T cells.

Background	Description	Vectors	Electroporation	Stimulation	Transduction and yield	Reference
PB transposon platform	Optimization of PB transposon platform for T-cells engineering using GFP as reporter	different quantities of pIRII-eGFP and pCMV-PB transposase	5x10^6 PBMCs using Nucleofector Device (Lonza, program U-014) with the human T-cell Nucleofector Kit	stimulation with CD3/CD28 mAbs and cytokines (IL-2, IL-15, IL-7, IL-4); transgenic T cells were selected on day 8 and expanded with feeder cells (autologous PBMCs or modified K562 cells)	Optimal results were obtained with 5µg of transposon and transposase with a transfection efficiency of 20%, improved to 30-40% with addition of IL-15.	Nakazawa et al., 2009 (120)
B-ALL	anti-CD19 CAR-T cells lacking the spacer IgG1 Fc region demonstrated superior efficacy in murine B-ALL xenograft models	pVAX1PB (5µg) + pVAX1SPBase (5µg)	4x10^6 PBMCs using Neon Electroporator with single pulse, 20 ms and 2400 V	Electroporated cells were cultivated in presence of IL-15 and stimulated with irradiated autologous PBMCs on D1 and after every 7 days.	Expansion: 100-fold after 22 days CAR expression: from 35% to 97%, depending on the construct	Bishop D. C. et al., 2018 (111)
CD19+ B-cell malignancies	Anti-CD19 CAR T cells manufactured in the presence of IL-4, IL-7 and IL-21 showed effective cytotoxic activity in vitro	5 µg (2:1 mixture of PB transposon vector and pCMV-PB hyperactive-transposase)	4x10^6 PBMCs using Neon electroporator inbuffer T (1x20 ms/2300V)	Electroporated cells were stimulated the day after in the presence of IL-4, IL-7 and IL-21 (stimulation by CD19 expressed on the surface of B cells in PBMC)	CAR expression: 90% in the presence of IL-4, IL-7 and IL-21. 30% when stimulated with anti-CD3/CD28 mAbs Expansion: from 4x10^6 to about 30-40×10^6 in 17 days and 100-120×10^6 in 24 days	P. Ptackova et al., 2018 (131)
CD19+ B-cell malignancies	anti-CD19 CAR T cells generated with co-electroporation of linear DNA transposon and mRNA encoding transposase showed lytic activity in vitro	pPB DNA linear transposon produced by PCR (3-0,3µg) + hyPBase mRNA transposase (12 µg) with 3′-O-Me-m7G (5′) PPP(5′) G RNA cap structure	1x10^7 PBMCs electroporated as in Ptackova et al., 2018 (131)	stimulation with TransAct reagent the day after electroporation and expansion for 21 days in the presence of IL-4, IL-7, and IL-21	CAR expression: 60-70% after 14-21 day of expansion Expansion: from 1x10^7 to 1x10^7 in 14 days and 1x10^8 in 21 days	I. Kastankova et al., 2021 (125)
Neuroblastoma	Anti-GD2 CAR T cells manufactured using autologous PBMCs pulsed with a pool of viral peptides showed effective antitumor response in xenograft model when combined with MEK inhibitor	pIRII-GD2-28Z CAR plasmid (7.5µg) + pCMV-PB transposase plasmid (7. 5µg)	2x10^7 PBMCs using 4D-Nucleofector and the P3 Primary Cell 4D-Nucleofector X kit, program FI-115 [See Morita D. et al., 2018 (121)]	stimulation with 5x10^6 autologous PBMCs pulsed with MACS PepTivator (AdV5 Hexon, HCMVpp65, EBNA-1, and BZLF), IL-7 and IL-15. Transfer to anti-CD3 or anti-CD28 mAb-coated plates on day 7 and expansion in G-Rex 6 Multi-Well Cell Culture Plates (Wilson Wolf Corporation, New Brighton) on day 9	CAR expression: 44% ± 6% at day 14 after transfection.	Tomida A. et al., 2021 (127)
HER2 positive solid tumor	HER2-CAR-T cells showed the ability to control Her2-positive tumor in mice	pIRII-HER2-28z plasmid (5µg) + pCMV-PB transposase plasmid (7.5µg)	20x10^6 PBMCs using 4D-Nucleofector and the P3 Primary Cell 4D-Nucleofector X kit, program FI-115 or the MaxCyte ATX protocol RTC 14-3.	Electroporated cells were stimuled with PBMC, electroporated with plasmid encoding tHER2, CD80 and 4-1BBL and UV-inactivated, on day 1 and cultivated in presence of IL-7 and IL-15 for 14 days	Expansion: 8 ± 1 fold CAR CAR expression: 60% ± 9% at day 14.	Nakamura K. Et al, 2021 (126)