1) Cell type selection
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Positive or negative selection of CD3+ and/or CD4+ or CD8+
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Easy separation by immunomagnetic beads;
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high cost Negative selection methods are inherently less pure than positive selection methods |
γδ T cells
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Possible to use in the allogeneic setting |
Few numbers of cells; laborious and expensive expansion |
EBV-CTLs
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Use in B cell malignancies and EBV-related infections |
Few numbers of cells |
NK cells
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Applicable for allogeneic settings, low toxicity effects, several sources |
Few numbers of cells; laborious expansion and difficult to genetically modify |
Monocytes/
Macrophages
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Applicable for allogeneic settings, low toxicity effects on pre-clinical models, potential application for solid tumors |
Laborious transfection and expansion procedures, no results available from clinical trials |
2) Activation
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Anti-CD3/CD28 beads/antibodies
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Efficient activation, easy handling |
Long cultures may induce terminally differentiated/exhausted T cells |
Cytokines
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IL-2: efficient for T cell expansion IL-7, IL-15, IL-21: induction of memory and/or Stem-like phenotype |
IL-2: high doses and long-term cultures may induce T cell exhaustion or expansion of regulatory CD4+ T cells, |
Stimulatory
cell lines
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Efficient activation, easy handling |
Risk of residual stimulatory cells in the final product, scale-up difficulties and licensing restrictions |
3) Gene delivery
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Viral-Based CAR delivery
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Best established techniques for transgene delivery. |
Risk of oncogene activation, high production cost, for clinical use (GMP) an extensive set of strict regulations should be followed |
Gamma Retroviral
Lentivirus
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Used in Yescarta and Tecartus Used in Kymriah, Breyanzi, Abecma and many other trials. Safer and better integration properties. Insert genetic material into non-dividing cells |
Oncogenic properties, high costs, Insertion only in dividing cells, It can be immunogenic Economic Costs, Risk of insertional mutagenesis, It can be immunogenic
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Non-viral-Based CAR delivery
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Easier to produce on a large scale, chemically characterize, greater reproducibility, greater transgenic capacity, fewer concerns about biosafety |
Standardization of techniques is more difficult compared to the use of viral vectors; High rates of cell death with nucleoporation; Lipofectamine-induced toxicity. |
Sleeping Beauty (
SB)
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Good results in T-cells, antitumor activity both in vitro and in vivo, SB100X was initiate to treat multiple myeloma patients |
Low integration rate in a large scale |
Piggy bac
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Properties more similar to viral-based vectors, also with CAR anti-CD73, MSLN, EGFRvIII, and PSMA to treat solid tumors. |
Two cases of malignant lymphoma derived from CAR gene modified T-cells prepared with PB vector were reported3939 Micklethwaite KP, Gowrishankar K, Gloss BS, Li Z, Street JA,Moezzi L, et al. Investigation of product derived lymphoma following infusion of piggyBac modified CD19 chimeric antigen receptor T-cells. Blood. 2021. https://doi.org/10.1182/blood.2021010858 . Epub ahead of print. PMID: 33974080. https://doi.org/10.1182/blood.2021010858...
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In vitro transcribed (IVT) mRNA
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Low risk of insertional mutagenesis |
transient expression of CAR |
Nanoplasmids
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Low risk of insertional mutagenesis, long-term transgene expression suitable for large scale production |
Difficult to produce |
4) Expansion
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T-Flasks
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Most affordable consumables |
Time consuming, handling from trained operators, contamination risk, no agitation |
Culture bag
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Low risk of contamination |
Handling from trained operators, no agitation |
G-Rex
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Less timing consuming from operators, superior final volume |
Handling from trained operators, no agitation |
Rocking motion bioreactor
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Closed system, low probability of contamination, cells kept in constant agitation, final volume up to 25 liters |
Not indicated for cells sensible to shear stress |