Table 10 Protocol features for G-rex systems
From: Lymphocyte expansion in bioreactors: upgrading adoptive cell therapy
| Â | 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 |