In-depth Characterization of a TCR-specific Tracer for Sensitive Detection of Tumor-directed Transgenic T Cells by Immuno-PET

Abstract

A number of different technologies have been developed to monitor <i>in vivo</i> the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using ⁸⁹Zr-Df-aTCRmu-F(ab')₂ targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth <i>in vitro</i> characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability <i>in vitro</i> and <i>in vivo</i>. Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells <i>in vivo</i>. Local application of ⁸⁹Zr-Df-aTCRmu-F(ab')₂-labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x10⁴ cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the ⁸⁹Zr-Df-aTCRmu-F(ab')₂ tracer, PET/CT imaging and subsequent <i>ex vivo</i> T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x10⁴ T cells. PET signals correlated well to total numbers of transgenic T cells detected <i>ex vivo</i> independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation.