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Table 10 Protocol features for G-rex systems

From: Lymphocyte expansion in bioreactors: upgrading adoptive cell therapy

  Protocol features Starting material Expansion Comparator Purity Functionality changes
Author (ref) - year IL-2 [IU/mL] Stimulation Medium Suppl. Source Seed [cells /mL] Vol. [mL] Culture system Fold Days System Fold days Target Cytotoxicity
T cell
Vera [158] - 2010 50 Irr. EBV-LCLs RPMI; CM 10% FBS; glutamine T cells from PBMC 5 × 105 30 G-rex40 1700–2200 23 plates 3–5 23 No change in phenotype; > 90% CD3+ cells (96.7 ± 1.7 vs 92.8 ± 5.6; G-rex vs 24-well), CD8+ (62.2% ± 38.3 vs 75% ± 21.7). Cytolytic activity maintained, killing vs EBV-LCL 62% ± 12 vs 57% ± 8;
G-rex vs 24-well plate
 Chakraborty [173] - 2013 50 Anti CD3/CD28, temsirolimus, Irr. APCs RPMI 10% AB, glutamine BME nTregs from PMBC NP 400 G-rex100 > 600 21 plates 5–6 21 Percentage of CD4 + CD25+ remained stable at 92 ± 5% by day 21 FoxP3+ cells increased from day 1 to day 21 of culture. No compromise in telomere length.
 Gerdemann [174] - 2013 NP Irr. nucleofected DCs, IL4 IL7 RPMI; CM 10% FBS; glutamine rCTLs from PBMC 2 × 107 30 G-rex10 7–28 9–11 NP NP NP CD3+ T cells (mean 98.6 ± 0.1%), CD8+ (59.6 ± 2.7%) CD4+ (34.1 ± 2.5%) CTL lines specific for EBV, CMV, and Adv antigens but not alloreactive.
 Ramanayake [175] - 2015 50 Irr. Autologous PBMCs or Nalm-6 cells;
IL15
AIMV 10% AB OR FBS T cells from PBMC 107 NP G-rex10 680.4–765.4 fold 22 plates 326.8–576.0 fold 22 57% CAR expression (range 50–63%) in G-rex10 vs. 66% (35–78%) in plates; CD3+ in G-rex10 88% vs. 96% in plates NP
Orio [160] - 2015 NP IL-4, IL-7, IL-21 RPMI; CM 10% AP, glutamine T cells from PBMC 2 × 107 NP G-rex10 9.2 12–14 NP NP NP Balanced CD4/CD8 T-cell ratio, slight CD8 predominance. Antigen-specific, allo-tolerant, anti-EBV T-cell lines
Jin [176] - 2018 3000 Irr. allogeneic PBMCs; anti-CD3 AIMV 5% AB, glutamine T cells from PBMC 107 800–4500 G-rex500 MCS 1890 ± 138 21 NP NP NP CD4+ T-cells were favored transduction efficiency of 74 ± 10% and 93 ± 1.4% Transduced T-cells lyse CASKi cells (48 ± 4%) and 293 cells expressing E6 (72 ± 1%). E6 and E7 TCR transduced T-cells produced IFN-γ and TNF-α.
Kuranda [177] - 2019 Used GM-CSF, IL-4, IL-1b, Flt3L, TLR8L, TNF-a, PGE2, IL-7, IL-2, IL-15, IL-7. AIMV 10% HS Ag or AdV5-reactive CD8+ from PBMC or CB 107 NP G-rex10 11 fold vs. std. 10 NP NP NP NP More CD8+ T cells producing TNF-α, IL-2, IFN-γ, and/or MIP-1b for G-rex cultures. IFN-γ, and MIP-1b were undetectable in plates.
Gagliardi [162] - 2019   Viral supern. and Vectofusin-1; IL7, IL15, antiCD3/CD28 TexMACS GMP 10% FBS Transd. T cells from PBMC NP 1000 G-rex100 MCS Higher than bags   Bags    No difference in CD45RA and CD62 L cell expression or CD4:CD8 ratio Transduction in G-rex 55 ± 7%, vs. 73 ± 7% in bag. More viable and transgenic cells from G rex. Secretion of cytokines did not differ.
Xiao [178]- 2018 300 Zometa; Irr K562 Clone A APCs; OKT3 AIMV 5% AB Vγ9Vδ2T cells from PBMC NP NP G-rex10 & 100 10,995 ± 3078 17 NP NP NP The Vγ9Vδ2T-cell purity achieved was 85.98% ± 10.28%.
The NK population decreased from 20 to 4.6%
G-rex promoted phenotypic changes: TEFF cell decreased from 66.6–51%; TEM cell increased from 21 to 37.6%. around 40–80% target cell killing.
NK cell
Lapteva [106] - 2012 10 Irr. K562-mbIL15-41BBL cells SCGM 10% FBS; glucose CD56+ CD3-NK cells from PBMC 2 × 106 400 G-rex100 442 ± 29 10 Bags 227 ± 91 10 Less than 35% proliferation of T cells was detected during the NK cell expansion. NP
Shah [179] - 2013 100 Irr. APC cells RPMI; CM 10% AB NK from Cryopreserved CB NP NP GP500 2389 14 NP NP NP 95% purity for NK cells (CD56+/CD32) and less than 1% CD3+ cells. Significant in vivo activity against MM in a xenogeneic mouse model
TIL
Jin [180] - 2012 3000 Irr. allogeneic PBMC, anti-CD3 AIMV 5% AB Tumor fragments or tumor digest cells 5 × 106 400 G-rex100 176 + 136 7 T175 167 ± 37 7 Phenotype similar to TIL produced with 24-well plates, T-175 flasks, and bags. IFN-γ similar to T-175 flasks and bags (Table 4).
Forget [181] - 2014 Used Anti CD3; Irr. allogeneic PBMC OR Aapc RPMI 10% AB, glutaminepyruvate, HEPES, BME Melanoma tumors 5 × 106 NP G-rex100 M > 2000 NP NP NP NP Mostly CD3+ cells for both conditions Decreased CD28 expression in T-flask system was not observed when using the G-rex flasks
Forget [159] - 2016 Used anti-CD3, Irr. allogeneic PBMC   NP Melanoma tumors 5 × 106 400 G-rex100 M 2653 14 Plate + bag 1210 14 G-rex did not favor clonal selection No Va or Vb expression was lost with any system. TCR diversity was not altered. The OCR of the TIL in G-rex almost tripled, demonstrating an enhanced mitochondrial capacity