Table 2.
Manufacturing protocols associated with preclinical studies employing SB in the context of CAR T cells.
| Background | Description | Vector | Electroporation | Stimulation | Transduction and Yield | Reference |
|---|---|---|---|---|---|---|
| B-ALL and AML | Anti-CD19 CAR-T cells showed proof-of-concept tumor eradication in B-ALL xenograft models; anti-CD123 CAR T cells controlled KG-1 AML in xenograft models | CD19CAR/pT MNDU3 transposon (15 μg) + pCMV‐SB11 transposase (5 μg) |
10^7 peripheral blood mononuclear cells (PBMCs) using 4D‐Nucleofector system (Program EO-115) with P3 Primary Cell 4D‐Nucleofector X kit (Lonza) |
Autologous PBMCs irradiated with 60Gy γ‐rays are added after electroporation and OKT‐3 is added at day 1. IL‐2 is added weekly. | CAR expression: 75.6% for CD123.CAR and 80% for CD19.CAR Large scale T cell expansion: 23-fold in 18-21 days CAR T cell expansion: from 60×10^6 to 8.6×10^8 |
Magnani et al; 2018 (44) |
| AML | Anti-CD33 CAR-T cells showed delaying AML progression in patient-derived xenograft models |
CD33CAR/pT transposon (15 μg) + SB100X-pT4 transposase (0.5 μg) |
As Magnani et al.;2016 (87) | As Magnani et al.;2016 (87) | CD33CAR expression: 63.7%T cell expansion: 38.8-fold after 3 weeks. | Rotiroti et al.;2020 (92) |
| Glioma | Production of EGFRvIII CART cells in two weeks showed superior therapeutic efficacy in mice bearing established orthotopic gliomas | EGFRvIIICAR pT/neo transposon (10 μg) + pCMV-SB11 transposase (5 μg) |
20 × 10^6 PBMCs or T cells using Amaxa Nucleofector 2B (Lonza) with program U-014 in T-cell electroporation buffer (Lonza) |
CAR T cells were stimulated with 100 Gy-irradiated EGFRvIII+ K562 ells in the presence of IL-21. After7 days, T cells were restimulated in the presence of IL-2 and IL-21. | CAR T cell (back calculated inferred numerical expansion): From 10^6 to around 5×10^7 in two weeks and 10^9 in 30 daysEGFRvIII CAR expression: around 90% | Caruso et al.;2019 (102) |
| CD19+ B-cell malignancies | Anti-CD19 CAR-T cells generated in 8 days showed effective antitumor response in mice xenografted with RS4;11 or Nalm-6 B-cell leukemias | pT3 19BBz CAR transposon (20 μg) + pCMV-SB100x transposase (1 μg) |
10^7 PBMC using Amaxa Nucleofector 2B with program U-14 | After electroporation cells are cultured with IL-2 and, 2h later, are activated with T Cell TransAct (Miltenyi Biotec) | Absolute number of T‐cell expansion: from 10^7 to 3.6×10^7 after 8 days of cultureCAR expression range: 20.4%–37.3% | Chicaybam et al.;2020 (94) |
| Lymphoma | anti-CD19 CAR T cells engineered with MC SB vectors eradicated lymphoma cells in Raji xenograft model | pT2 CAR EGFRt MC DNA (1 μg) +pCMV-SB100X MC (1:1 ratio) or pCMV-SB100X mRNA (1:4 ratio) | T cells are activated with anti-CD3/CD28 beads (Thermo Fisher Scientific) and on day 2 electroporated (1×10^6 T cells) using 4D-Nucleofector | After electroporation T cells are propagated with IL-2. Prior to functional testing, EGFRt-positive T cells are enriched and expanded with irradiated CD19+ feeder cells for at least 7 days | CAR T cell expansion: From 1×10^6 to around 20×10^6 (MC-MC) and 12×10^6 (MC-mRNA) in 2 weeksCAR expression: 49.8% for MC-MC and about 40% for MC-mRNA on day14 | Monetzi et al;2017 (81) |