CAR T-cells have shown limited applications against solid tumours due to low solid target trafficking, thus limiting efficacy¹. Direct PET cell tracking can provide information to help tackle these issues, by delivering quantifiable and non-invasive CAR T-cell biodistribution data². In this study, we determine radiolabelling tolerance on viability and functionality of novel NKG2D ligand targeting CAR T-cells, perform PET tracking of these radiolabelled CAR-T cells in NSG mice bearing head and neck cancer tumours and assess the impact of external irradiation upon tumour T-cell infiltration.

[⁸⁹Zr]Zr(oxinate)₄, synthesised with high radiochemical purity using a kit formulation^3,4, labelled GMP-grade LEU011 CAR T-cells at varying concentrations to determine maximum viable tolerated radioactivity (Fig 1A). NSG mice, subcutaneously implanted with 7.5 x 10⁶ ffluc-HN3 head and neck cancer cells, were monitored via IVIS imaging and callipers. Tumour-bearing mice were grouped into five imaging/treatment sets (Fig 1B). Milestone A assessed the therapeutic potential of unlabelled CAR T-cells (~15 x 10⁶ cells). Milestone B1 examined the spatiotemporal biodistribution and efficacy of ⁸⁹Zr-CAR T-cells and contrasted their therapeutic potential with unlabelled cells (~15 x 10⁶ cells, 20 kBq/10⁶ cells). Milestone B2 explored the impact of DFO administration on removing unchelated ⁸⁹Zr released from labelled cells in vivo (~15 x 10⁶ cells, 20 kBq/10⁶ cells).   Milestone C explored the impact of tumour-targeted irradiation (~8 Gy) upon tumour infiltration, biodistribution, and efficacy of the ⁸⁹Zr-CAR T-cells (~15 x 10⁶ cells, 20 kBq/10⁶ cells). Immunostaining of tumour and spleen tissues from different groups was performed to confirm CAR T-cells’ proliferation and therapeutic efficacy.

The viability of ⁸⁹Zr-CAR T-cells post-labelling with 20 kBq/10⁶ cells ranged from 80 ± 1 % (24 h) to 16 ± 6 % (7 d), similar to unlabelled CAR T-cells. Concurrently, ⁸⁹Zr retention decreased from 40 ± 1 % at 24 h to 12 ±1 % at 7 d post-labelling which is in line with previous ⁸⁹Zr-oxine labelled-T cell studies^4,5. LEU011 CAR T-cells inhibited HN3 tumour growth compared to the placebo group (Fig 1C). In vivo ⁸⁹Zr-CAR T-cell tracking revealed sequential localisation in the lungs, followed by accumulation in the liver, spleen, and tumour, with peak tumour accumulation at 24h (Fig 1D,G). Milestone B2 demonstrated the capability of DFO to remove ⁸⁹Zr from relevant organs. ⁸⁹Zr removal by DFO can be relevant as a clinical intervention post-imaging to remove radiation burden (Fig 1D,G). Pretherapy tumour irradiation enhanced tumour infiltration of ⁸⁹Zr-CAR T-cells >2-fold compared to the non-irradiated group (Fig 1D,G). Zr labelling had minimal impact on the therapeutic effect of CAR T-cells (Fig 1E,F).

Novel CAR T-cells labelled with ⁸⁹Zr-oxine exhibited comparable viability and therapeutic efficacy to the non-radiolabelled controls. Radiolabelling enabled in vivo tracking of a therapeutic CAR T-cell dose in HN3 tumour-bearing mice. Imaging indicated CAR T-cell accumulation in tumours, which was enhanced by greater than 2-fold after external beam irradiation in solid tumours. The immunological staining results will further confirm the therapeutic effect of CAR T-cells and increased cell infiltration due to tumour irradiation.  

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Figure 1. In vivo cell tracking of ⁸⁹Zr-CAR-T cell (LEU-011) in an NSG mice model of subcutaneous head and neck cancer HN3 cell line: (A) Cell labelling protocol to determine the impact of radioactivity on cell viability; (B) Different in vivo study groups for therapeutic and imaging study in HN3 subcutaneous model in NSG mice; (C) Tumour monitoring for the therapeutic study showing tumour volume percentage change. The therapy group showed decrease in tumour volume compared to the placebo group which showed a continuous increase in tumour volume;  (D) Maximum intensity projections of sagittal planes of images from three study groups at t=24 hr with tumour highlighted in the dashed lines. Images show decreased Zr accumulation in bones and tumour for DFO administered groups while increased tumour accumulation in the tumour irradiated (EBRT group) compared to non-DFO and non-irradiation group; (E) Therapeutic effect of ⁸⁹Zr-CAR-T cells;(F)Combined therapeutic effect of irradiation and ⁸⁹Zr-CAR-T cells; (G) Tumour accumulation of ⁸⁹Zr-CAR-T cells over multiple days and multiple groups showing higher accumulation in EBRT group compared to other study groups.

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