Spatio-temporal biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells by PET imaging in a preclinical tumor model - PubMed (original) (raw)
Spatio-temporal biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells by PET imaging in a preclinical tumor model
Naomi S Sta Maria et al. Sci Rep. 2021.
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.
© 2021. The Author(s).
Conflict of interest statement
The University of Southern California, Dr. Peisheng Hu, and Dr. Alan L. Epstein are equity holders in Cell BT, Inc. All other authors declare that there is no conflict of interest.
Figures
Figure 1
Study timelines. (a) In vitro measurements of cell health and function of 89Zr labeled and unlabeled non-transduced (mock) T-cells and CAR T-cells were obtained in a separate experimental trial. Percent lysis of target Raji cells (cytotoxicity) and released cytokine levels in situ by the effector 89Zr-oxine labeled and unlabeled mock and CAR T-cells were measured 24 h after incubation of the effector cells with the target cells. Incubation of effector and target cells began 3 h post 89Zr-oxine labeling. Aliquots of 89Zr-oxine labeled and unlabeled mock and CAR T-cells were subcultured for in vitro viability and live cell number counts on the same in vivo scan timepoints. (b) In vivo biodistribution measurement of 89Zr-oxine labeled CAR T-cells began with CAR T-cell preparation and inoculation of NSG mice with Raji cells 12 days prior 89Zr-oxine labeling and IV administration. NSG mice were randomized to receive either a low (126.7 ± 2.5 kBq, 1.87 ± 0.04 × 106 cells, n = 3 mice), middle (542.6 ± 92.1 kBq, 7.14 ± 0.45 × 106 cells, n = 5 mice), or high (1491.7 ± 36.1 kBq, 16.83 ± 0.41 × 106 cells, n = 3 mice) dose of 89Zr-oxine labeled huLym-1-BB3z-CAR T-cells and underwent PET/MRI scans on day 0, 1, 2, and 5 time points, which corresponded to 3, 24, 48, and 120 h after CAR T-cell injection. Activity concentrations were obtained from PET scans and expressed as percent injected dose per gram of tissue of manually defined regions of interests that were identified on T1-weighted MRI anatomical scans. Coplanar PET and MRI scans were acquired simultaneously and were registered to the same space. Invasive ex vivo biodistribution was performed on day 6 post injection to complement the in vivo PET imaging study arm. 89Zr-oxine labeled and unlabeled CAR T-cells were subcultured for in vitro viability and live cell number counts on the same scan timepoints.
Figure 2
89Zr-oxine labeling does not significantly alter cell health and function of huLym-1-A-BB3z-CAR T-cells. (a) Schematic of 2nd generation CAR construct used for huLym-1-A-BB3z-CAR T-cells. Adapted from Zheng, et al., 2020 with permission of the copyright owner. (b, c) Flow cytometry analysis of CAR expression on non-transduced (mock) or transduced primary human T-cells (huLym-1-A-BB3z-CAR T-cells) on day 7. CAR expression was measured using Dylight 650-conjugated antibody against the 261 tag. (d, e) Percent viability measurement of subcultured 89Zr-oxine labeled and unlabeled mock and huLym-1-A-BB3z-CAR T-cells on day 0, 1, 2, and 4 time points, which correspond to 3, 24, 48, 120 h after 89Zr-oxine labeling, respectively. (f, g) Total live cell count (× 106) of subcultured 89Zr-oxine labeled and unlabeled mock and CAR T-cells on the same time points. Viability and live cell number plots show dash lines as means, colored bars as standard deviation ranges, and open circles as individual data points. Significant differences are shown with p-values above brackets. A value of p < 0.05 was used to indicate statistical significance. (h) Cytotoxicity of 89Zr-oxine labeled and unlabeled mock and huLym-1-A-BB3z-CAR T-cells against Lym-1-positive Raji cells. (i, j) IL-2 and IFN-γ cytokines released by 89Zr-oxine labeled and unlabeled mock and huLym-1-A-BB3z-CAR T-cells. Cytotoxicity and cytokine release plots show means (closed circles) and standard deviation error bars. Labeling with 89Zr-oxine show unaltered CAR T-cell cytotoxicity and IL-2 and IFN-γ cytokine release.
Figure 3
Representative PET/MRI of 89Zr-oxine labeled huym-1-A-BB3z-CAR T-cells. (a) Central figure shows a representative coronal PET (colored: nih fire) and MRI (gray) overlay image of a Raji-bearing NSG mouse 24 h after IV administration of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells (1517 kBq, 16.4 × 106 cells). Mouse image on the lower left shows orientation of coronal slice. Medial columns of transverse cross-sections show PET/MRI overlay images with slice positions indicated along the central coronal PET/MRI image. Lateral columns of transverse cross-sections show the same MRI slices with manually defined regions of interests (ROI) in color within the delineated organ or tissue boundaries in dashed lines. ROIs analyzed were the lungs (green), left ventricle for blood (dark magenta), liver (cyan), spleen (magenta), brain (red), tumor (dark green), and bone (yellow). ROI activity concentrations were obtained from PET images and expressed as percent injected dose per gram of tissue for CAR T-cell biodistribution analyses. Scale bars indicate 10 mm for both coronal (white) and transverse images (yellow). Color bar indicates PET activity concentration in × 10–3 kBq/mm3 units. (b) Representative coronal PET/MRI slices at the 3, 24, 48, and 120 h timepoints following IV injection of huLym-1-A-BB3z CAR T-cells in same mouse from (a). Mouse on the lower left shows orientation of coronal images. Activity was observed highest in the lungs at 3 h and diminished overtime, while activities in liver and spleen increased at the later time points. Activity accumulation in tumor was evident by 24 h following 89Zr-oxine labeled CAR T-cell injection. Color bar indicates PET activity concentration in × 10–3 kBq/mm3 units. White scale bar on lower right indicates 10 mm.
Figure 4
Tissue biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells dose escalation. Significant changes in %ID/g overtime were observed in all regions. (a–g) In vivo blood, bone, brain, liver, lungs, spleen, and tumor %ID/g of Raji-bearing NSG mice administered with low (mean ± SD: 126.7 ± 2.5 kBq, 1.87 ± 0.04 × 106 cells), middle (542,6 ± 92.1 kBq, 7.14 ± 0.45 × 106 cells) or high dose (1491.7 ± 36.1 kBq, 16.83 ± 0.41 × 106 cells) of 89Zr labeled huLym-1-A-BB3z-CAR T-cells (Low dose, n = 3 mice; Middle dose, n = 5 mice; High dose, n = 3 mice). Columns with error bars indicate means and standard deviations (SD) and open circles as individual data points. (h) Tumor-to-blood ratio obtained by dividing tumor %ID/g by blood %ID/g to account for cell signal accumulation in extravascular tumor tissue. Significant cell accumulation was observed by beginning on day 1 to day 5 in NSG mice that received the middle dose, and in mice that received low dose on day 2, by one-sample t-test vs. 0.6 threshold to overcome the enhanced permeability and retention (EPR) effect. (i) Ex vivo biodistribution %ID/g of low, middle, and high CAR T-cell doses on day 6 post CAR T-cell injection. Columns and error bars indicate means and SD, respectively. Statistically significant findings between comparisons are indicated with p-values above brackets. A value of p < 0.05 was used to indicate statistical significance.
Figure 5
Parallel in vitro viability and live cell count of subcultured 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells administered in mice. (a) Percent viability and (b) total live cell count (× 106) of subcultured 89Zr-oxine labeled and unlabeled CAR T-cells from the same cell batch administered to live mice. Viability and live cell count plots show dash lines as means, colored bars as SD, and open circles as individual data points. Statistically significant findings between comparisons are indicated with p values above brackets. A value of p < 0.05 was used to indicate statistical significance.
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