Table 2

Methods and tools to assess the expression of CARs and TCRs on engineered T cells

LevelMethodMeasurementObservations
GenomicqPCRTCR/CAR vector copy numberMultiplexing; high throughput; cannot discriminate subtle VCN differences
ddPCRMultiplexing; high throughput; costly; more precise and sensitive than qPCR
ISSites of vector integrationSensitivity in assaying rarer clones; abundance of each transduced T cells can be bioinformatically inferred from IS data
TCR-seqPresence of the transgenic TCRDefines the T-cell clonal composition of the infused product; protocol optimization needed to detect codon optimized TCR sequences
TranscriptomicRNA-seqCAR/TCR mRNA abundanceCAR/TCR mRNA quantity depends on chromatin architecture, viral promoter, regulatory elements
Single-cell RNA-seqCAR/TCR mRNA quantity depends on chromatin architecture, viral promoter, regulatory elements; coupled to multimers for proteomic and transcriptomic evaluation; multiplexing; costly
TCR-seqTCR mRNA abundanceDefines the T cell clonal composition of the infused product; protocol optimization needed to detect codon optimized TCR sequences
RNAscope ISHCAR/TCR RNA expression in tissuesUsed 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 cytometryAnti-IgG Ab Protein LCAR expression on T-cell surfaceOptimized, 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 AbHigh 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 tagsCan 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 cellsAllows tracking of engineered T cells in patients and mice; no information on CAR/TCR expression on the T-cell membrane
pHLA multimersTCR expression on T-cell surfacePossible 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 antibodiesOverestimation 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 AbUseful when murinized TCR constant regions are used; possibility to measure with imaging if Ab is coupled to specific reporter molecules
In vivo imagingNuclear medicine imagingCapture biodistribution and expansion of engineered T-cellsPET, SPECT. Radio-labeled probes; multiplexing; negative effects of radiotracers on cell function
MRINon-invasive, semi-quantitative, high-resolution whole-body tracing of the T-cell product
Optical imagingUse of luciferase/substrate pairs. Widely available. Not used in the clinic.
Two photon microscopyCan 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.