Abstract
Quantitative in vivo monitoring of cell biodistribution offers assessment of treatment efficacy in real-time and can provide guidance for further optimization of chimeric antigen receptor (CAR) modified cell therapy. We evaluated the utility of a non-invasive, serial 89Zr-oxine PET imaging to assess optimal dosing for huLym-1-A-BB3z-CAR T-cell directed to Lym-1-positive Raji lymphoma xenograft in NOD Scid-IL2Rgammanull (NSG) mice. In vitro experiments showed no detrimental effects in cell health and function following 89Zr-oxine labeling. In vivo experiments employed simultaneous PET/MRI of Raji-bearing NSG mice on day 0 (3 h), 1, 2, and 5 after intravenous administration of low (1.87 ± 0.04 × 106 cells), middle (7.14 ± 0.45 × 106 cells), or high (16.83 ± 0.41 × 106 cells) cell dose. Biodistribution (%ID/g) in regions of interests defined over T1-weighted MRI, such as blood, bone, brain, liver, lungs, spleen, and tumor, were analyzed from PET images. Escalating doses of CAR T-cells resulted in dose-dependent %ID/g biodistributions in all regions. Middle and High dose groups showed significantly higher tumor %ID/g compared to Low dose group on day 2. Tumor-to-blood ratios showed the enhanced extravascular tumor uptake by day 2 in the Low dose group, while the Middle dose showed significant tumor accumulation starting on day 1 up to day 5. From these data obtained over time, it is apparent that intravenously administered CAR T-cells become trapped in the lung for 3–5 h and then migrate to the liver and spleen for up to 2–3 days. This surprising biodistribution data may be responsible for the inactivation of these cells before targeting solid tumors. Ex vivo biodistributions confirmed in vivo PET-derived biodistributions. According to these studies, we conclude that in vivo serial PET imaging with 89Zr-oxine labeled CAR T-cells provides real-time monitoring of biodistributions crucial for interpreting efficacy and guiding treatment in patient care.
Highlights
Chimeric antigen receptor (CAR) T-cells express genetically modified receptors or antibodies that recognize and bind tumor antigens and trigger a tumor cell killing c ascade[1]
We investigated whether, over time, the amount of adoptively transferred chimeric antigen receptor (CAR) T-cells correlated with accumulation at the tumor target and at off-target sites, such as liver, spleen, bone and brain, and provide information about the efficacy of the immunotherapy dose
To determine that 89Zr-oxine labeling had no detrimental effect on cell health, we subcultured 89Zr-oxine labeled and unlabeled mock and huLym-1-ABB3z-CAR T-cells for percent viability (%viability) and total live cell number measurements over the same time points as the in vivo imaging. 89Zr-oxine labeled mock T-cells showed significantly less %viability compared to unlabeled mock T-cells on day 2 (p = 0.0111) and 5 (p = 0.0002) post labeling (Fig. 2d), while %viability was not significantly different between 89Zr-oxine labeled and unlabeled CAR T-cells (Fig. 2e)
Summary
Chimeric antigen receptor (CAR) T-cells express genetically modified receptors or antibodies that recognize and bind tumor antigens and trigger a tumor cell killing c ascade[1]. Measurements of 89Zr-oxine cell specific activity show that the complex remains intracellularly This process is analogous to indium, which favors an oxidation state of 3+, forming InL3 complexes and is currently clinically available as 111InL3. While previous studies demonstrated feasibility of 89Zr labeling of their cells of interest and accumulation at their target, these studies showed off-target accumulation of the radiolabeled cells after adoptive transfer in vivo In this experiment, we investigated whether, over time, the amount of adoptively transferred CAR T-cells correlated with accumulation at the tumor target and at off-target sites, such as liver, spleen, bone and brain, and provide information about the efficacy of the immunotherapy dose. NSG mice were inoculated with non-Hodgkin Raji lymphoma cells subcutaneously and were intravenously (IV) administered different doses of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells. Invasive ex vivo biodistribution was performed as an endpoint to complement the in vivo PET imaging study arm
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