Table 1.

Methods and tools used for toxicity assessment of engineered T cells, their advantages, limitations, and potential improvements

Toxicity assessment	Methods and tools	Advantages	Limitations	Required improvements
CRS and neurotoxicity	Ex vivo coculture models (eg, CAR T cells, tumor cells and macrophages)	Ease of implementation. Suitable for mechanistic insights.	Not always correlated with in vivo CRS. Do not recapitulate the in vivo complexity.	Integration of other cells. Development of human 3D models (organoids, organotypical, organ-on-a-chip).
	Serum biomarkers from CAR T cell-treated patients.	Very good correlation of certain biomarkers with future development of severe CRS and neurotoxicity.	Predefined biomarkers	Unbiased multiparametric assessments (longitudinal, spatial, single cells)
	Syngeneic models	Mimic the crosstalk between CAR T cells and innate immune cells well	Use of mouse CAR constructs. Differences between mouse and human T cells (poor persistence of murine T cells).	Improvement of CAR engineering in murine T cells
	Immunocompromised SCID-beige mice	Partly functional innate immune cells. Partially correlated with patient CRS.	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	Complicated and long to set up. Variability in the human reconstitution. High costs. Long engraftment times. Time-consuming.	Model simplification and standardization. Model set-up with different tumor types. Improved characterization of neurotoxicity.
	Primates	Closely recapitulates patient CRS and neurotoxicity. High predictive value due to similar physiology between human and non-human primate cells.	Ethical considerations. High costs. Limited cohorts. Non-tumor bearing.	Increased accessibility and dedicated personnel
On-target and off-target off-tumor	NSG mice	Valuable when the expression of the target antigen is similar between human and murine cells
	Syngeneic models	Valuable when the expression of the target antigen is similar between human and murine cells. Generation of transgenic mice expressing the target antigen.	Intrinsic species- specific differences in terms of target expression. Generation of transgenic mice for multiple antigens is a laborious task.	Improvement of CAR engineering in murine T cells
	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	Laborious High costs. Long engraftment times. Time-consuming.	Model simplification. Model standardization. Model set-up with different tumor types.
	Primate models	High predictive values due to species-specific similarities	Ethical considerations. High costs. Non-tumor bearing. Limited cohorts.	Increased accessibility and dedicated personnel
	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)	High cost. Need to broaden the protein coverage.	Extension of the technology within the plasma membrane proteome and the secretome
	Target antigen expression measurement (via immunohistochemistry (IHC) staining)	Ease of implementation	Often predefined to certain tissues. Lack sensitivity. Limited to a few markers.	Functional two-densional and three-dimensional human models (organoid and organotypical) testing the responsiveness of CAR T cells against healthy tissues. Multiplex imaging approaches.
GVHD/rejection	MHC-disparate allogeneic mouse models		Impossible to test human T-cell products
	NSG mice	Xeno reactions can be used as a surrogate for GVHD with human CAR T cells. Coinfusion of CAR T cells and HLA mismatched PBMCs can be used to determine alloreactivity.	Not relevant to evaluate the rejection of infused CAR T cells. The need to use PBMCs derived from individuals with several HLA types (classes I and II and minor HLAs) might limit the utility of this in vivo model.
	Humanized NSG/SGM3 mice (reconstituted with human PBMCs or CD34+ cells)	Recapitulates the reactivity of human CAR T cells against allogeneic CD34+-derived cells. Can be used to study rejection of infused CAR T cells.	Complicated and long to set up. High costs. Long engraftment times. Time-consuming.
	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.