Toxicity assessment | Methods and tools | Advantages | Limitations | Required improvements |
CRS and neurotoxicity | Ex vivo coculture models (eg, CAR T cells, tumor cells and macrophages) |
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Serum biomarkers from CAR T cell-treated patients. |
| Predefined biomarkers | Unbiased multiparametric assessments (longitudinal, spatial, single cells) | |
Syngeneic models | Mimic the crosstalk between CAR T cells and innate immune cells well |
| Improvement of CAR engineering in murine T cells | |
Immunocompromised SCID-beige mice |
| Species-specific barriers requiring high amount of human CAR T cells and tumor cells | Model set-up with different tumor types | |
Humanized NSG/SGM3 mice (reconstituted with human PBMCs or CD34+ cells) | Recapitulate patient CRS and neurotoxicity well |
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Primates |
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| Increased accessibility and dedicated personnel | |
On-target and off-target off-tumor | NSG mice |
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Syngeneic models |
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Humanized NSG/SGM3 mice (reconstituted with human PBMCs or CD34+ cells) | Very valuable when the expression of the target antigen is limited to the hematopoietic compartment |
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Primate models | High predictive values due to species-specific similarities |
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Off-target screening with human cell microarray platform | Can evaluate off-target binding of human CAR T-cell therapy products (whole cells or scFv formats) |
| Extension of the technology within the plasma membrane proteome and the secretome | |
Target antigen expression measurement (via immunohistochemistry (IHC) staining) | Ease of implementation |
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GVHD/rejection | MHC-disparate allogeneic mouse models | Impossible to test human T-cell products | ||
NSG mice |
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Humanized NSG/SGM3 mice (reconstituted with human PBMCs or CD34+ cells) |
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Mixed lmphocyte reaction | Ease of implementation | Limited guidance on what HLA types (major and minor) on donor PBMCs must be tested in the assay | ||
TCR cross-reactivity | In vitro screening of TCR T-cell responses against human cell lines expressing diverse HLA alleles | Ease of implementation | ||
In vitro screening of TCR T-cell responses against mutated cognate peptides (positional scanning peptide libraries) | Predicts the peptide residues that are essential for TCR binding well | |||
Insertional mutagenesis and clonal dominance | In vitro cell-free assays | Ease of implementation in test tube | False positives | |
Ex vivo | Closer to reality because it assays effects in living cells | Often relies on surrogate cell lines | Perform assay in therapeutically relevant primary cells | |
In silico approach | Automated application through computer interface | False negatives |
CAR, chimeric antigen receptor; CRS, cytokine release syndrome; GVHD, graft-versus-host disease; scFv, single-chain fragment variable.