Level | Method | Measurement | Observations |
Genomic | qPCR | TCR/CAR vector copy number | Multiplexing; high throughput; cannot discriminate subtle VCN differences |
ddPCR | Multiplexing; high throughput; costly; more precise and sensitive than qPCR | ||
IS | Sites of vector integration | Sensitivity in assaying rarer clones; abundance of each transduced T cells can be bioinformatically inferred from IS data | |
TCR-seq | Presence of the transgenic TCR | Defines the T-cell clonal composition of the infused product; protocol optimization needed to detect codon optimized TCR sequences | |
Transcriptomic | RNA-seq | CAR/TCR mRNA abundance | CAR/TCR mRNA quantity depends on chromatin architecture, viral promoter, regulatory elements |
Single-cell RNA-seq | CAR/TCR mRNA quantity depends on chromatin architecture, viral promoter, regulatory elements; coupled to multimers for proteomic and transcriptomic evaluation; multiplexing; costly | ||
TCR-seq | TCR mRNA abundance | Defines the T cell clonal composition of the infused product; protocol optimization needed to detect codon optimized TCR sequences | |
RNAscope ISH | CAR/TCR RNA expression in tissues | Used in FFPE and frozen tissues; co-localization with multiple RNA transcripts and/or protein markers; spatial variation of the expression patterns in tissues. No information on whether CAR/TCR are present at the T-cell membrane. | |
Proteomic/flow cytometry | Anti-IgG Ab Protein L | CAR expression on T-cell surface | Optimized, affordable reagents; multi-step staining is required to avoid cross-reaction with IgG-like proteins; cannot independently stain different CARs on a dual-CAR expressing cell. |
Recombinant antigen-Fc proteins; anti-idiotype Ab | High specificity for CAR: can independently stain different CARs on dual-CAR expressing cells; for recombinant proteins, interference between antigen and detection method is a potential drawback after Ag recognition | ||
Ab against linkers or tags | Can be used in combination with other cell surface Ab in a one-step staining; Ab against linkers are typically not accessible outside industry; tags may influence CAR functionality due to structural changes | ||
Expression of gene reporters (EGFR, CD20) | Detection of genetically modified T cells | Allows tracking of engineered T cells in patients and mice; no information on CAR/TCR expression on the T-cell membrane | |
pHLA multimers | TCR expression on T-cell surface | Possible underestimation of the Td T cell population due to a more reliable binding of multimers to the CD8 transduced T cells; coupled to scRNAseq for proteomic and transcriptomic evaluation | |
TCR V antibodies | Overestimation of Td T cells due to the contaminant derived by the endogenous repertoire in ex-vivo samples; in the pre-infusion product quantify TCR expression in TCR gene edited cells | ||
Murine constant Ab | Useful when murinized TCR constant regions are used; possibility to measure with imaging if Ab is coupled to specific reporter molecules | ||
In vivo imaging | Nuclear medicine imaging | Capture biodistribution and expansion of engineered T-cells | PET, SPECT. Radio-labeled probes; multiplexing; negative effects of radiotracers on cell function |
MRI | Non-invasive, semi-quantitative, high-resolution whole-body tracing of the T-cell product | ||
Optical imaging | Use of luciferase/substrate pairs. Widely available. Not used in the clinic. | ||
Two photon microscopy | Can track engineered cells at the single cell level; increased spatial resolution; reduced photobleaching. |
Ab, antibody; ddPCR, digital droplet PCR; FFPE, formalin-fixed paraffin-embedded; IS, integration site; ISH, in situ hybridization; PET, positron emission tomography; qPCR, quantitative PCR; sc, single cell; SPECT, single photon emission CT; Td, transduced; V, variable region of the TCR; VCN, vector copy number.