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Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging.

ABSTRACT: INTRODUCTION:Chimeric antigen receptor (CAR) T-cells have been recently developed and are producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo 89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET). RESULTS:The 89Zr-DFO radiolabeling efficiency of Jurkat/CAR and human peripheral blood mononuclear cells (hPBMC)/CAR T-cells was 70%-79%, and cell radiolabeling activity was 98.1-103.6 kBq/106 cells. Cell viability after radiolabeling was >95%. Cell proliferation was not significantly different during the early period after radiolabeling, compared with unlabeled cells; however, the proliferative capacity decreased over time (day 7 after labeling). IL-2 or IFN-? secretion was not significantly different between unlabeled and labeled CAR T-cells. PET/magnetic resonance imaging in the xenograft model showed that most of the 89Zr-DFO-labeled Jurkat/CAR T-cells were distributed in the lung (24.4% ± 3.4%ID) and liver (22.9% ± 5.6%ID) by one hour after injection. The cells gradually migrated from the lung to the liver and spleen by day 1, and remained stable in these sites until day 7 (on day 7: lung 3.9% ± 0.3%ID, liver 36.4% ± 2.7%ID, spleen 1.4% ± 0.3%ID). No significant accumulation of labeled cells was identified in tumors. A similar pattern was observed in ex vivo biodistributions on day 7 (lung 3.0% ± 1.0%ID, liver 19.8% ± 2.2%ID, spleen 2.3% ± 1.7%ID). 89Zr-DFO-labeled hPBMC/CAR T-cells showed a similar distribution, compared with Jurkat/CAR T-cells, on serial PET images. CAR T cell distribution was cross-confirmed by flow cytometry, Alu polymerase chain reaction, and immunohistochemistry. CONCLUSION:Real-time in vivo cell trafficking is feasible using PET imaging of 89Zr-DFO-labeled CAR T-cells. This can be used to investigate cellular kinetics, initial in vivo biodistribution, and safety profiles in future CAR T-cell development.

SUBMITTER: Lee SH

PROVIDER: S-EPMC6946129 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging.

Lee Suk Hyun SH Soh Hyunsu H Chung Jin Hwa JH Cho Eun Hye EH Lee Sang Ju SJ Ju Ji-Min JM Sheen Joong Hyuk JH Kim Hyori H Oh Seung Jun SJ Lee Sang-Jin SJ Chung Junho J Choi Kyungho K Kim Seog-Young SY Ryu Jin-Sook JS

PloS one 20200107 1

<h4>Introduction</h4>Chimeric antigen receptor (CAR) T-cells have been recently developed and are producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo 89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET).<h4>Results</h4>The 89Zr-DFO radiolabeling efficie ...[more]

PMID: 31910217

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Project description:(89)Zr (half-life, 78.41 h) is a positron-emitting radionuclide that displays excellent potential for use in the design and synthesis of radioimmunoconjugates for immunoPET. In the current study, we report the preparation of (89)Zr-desferrioxamine B (DFO)-J591, a novel (89)Zr-labeled monoclonal antibody (mAb) construct for targeted immunoPET and quantification of prostate-specific membrane antigen (PSMA) expression in vivo.The in vivo behavior of (89)Zr-chloride, (89)Zr-oxalate, and (89)Zr-DFO was studied using PET. High-level computational studies using density functional theory calculations have been used to investigate the electronic structure of (89)Zr-DFO and probe the nature of the complex in aqueous conditions. (89)Zr-DFO-J591 was characterized both in vitro and in vivo. ImmunoPET in male athymic nu/nu mice bearing subcutaneous LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors was conducted. The change in (89)Zr-DFO-J591 tissue uptake in response to high- and low-specific-activity formulations in the 2 tumor models was measured using acute biodistribution studies and immunoPET.The basic characterization of 3 important reagents-(89)Zr-chloride, (89)Zr-oxalate, and the complex (89)Zr-DFO-demonstrated that the nature of the (89)Zr species dramatically affects the biodistribution and pharmacokinetics. Density functional theory calculations provide a rationale for the observed high in vivo stability of (89)Zr-DFO-labeled mAbs and suggest that in aqueous conditions, (89)Zr-DFO forms a thermodynamically stable, 8-coordinate complex by coordination of 2 water molecules. (89)Zr-DFO-J591 was produced in high radiochemical yield (>77%) and purity (>99%), with a specific activity of 181.7 +/- 1.1 MBq/mg (4.91 +/- 0.03 mCi/mg). In vitro assays demonstrated that (89)Zr-DFO-J591 had an initial immunoreactive fraction of 0.95 +/- 0.03 and remained active for up to 7 d. In vivo biodistribution experiments revealed high, target-specific uptake of (89)Zr-DFO-J591 in LNCaP tumors after 24, 48, 96, and 144 h (34.4 +/- 3.2 percentage injected dose per gram [%ID/g], 38.0 +/- 6.2 %ID/g, 40.4 +/- 4.8 %ID/g, and 45.8 +/- 3.2 %ID/g, respectively). ImmunoPET studies also showed that (89)Zr-DFO-J591 provides excellent image contrast, with tumor-to-muscle ratios greater than 20, for the delineation of LNCaP xenografts between 48 and 144 h after administration.These studies demonstrate that (89)Zr-DFO-labeled mAbs show exceptional promise as radiotracers for immunoPET of human cancers. (89)Zr-DFO-J591 displays high tumor-to-background tissue contrast in immunoPET and can be used to delineate and quantify PSMA-positive prostate tumors in vivo.

Project description:UNLABELLED:Tumor-associated macrophages (TAMs) are increasingly investigated in cancer immunology and are considered a promising target for better and tailored treatment of malignant growth. Although TAMs also have high diagnostic and prognostic value, TAM imaging still remains largely unexplored. Here, we describe the development of reconstituted high-density lipoprotein (rHDL)-facilitated TAM PET imaging in a breast cancer model. METHODS:Radiolabeled rHDL nanoparticles incorporating the long-lived positron-emitting nuclide (89)Zr were developed using 2 different approaches. The nanoparticles were composed of phospholipids and apolipoprotein A-I (apoA-I) in a 2.5:1 weight ratio. (89)Zr was complexed with deferoxamine (also known as desferrioxamine B, desferoxamine B), conjugated either to a phospholipid or to apoA-I to generate (89)Zr-PL-HDL and (89)Zr-AI-HDL, respectively. In vivo evaluation was performed in an orthotopic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo histologic analysis of tumor tissues to assess regional distribution of (89)Zr radioactivity was also performed. Fluorescent analogs of the radiolabeled agents were used to determine cell-targeting specificity using flow cytometry. RESULTS:The phospholipid- and apoA-I-labeled rHDL were produced at 79% ± 13% (n = 6) and 94% ± 6% (n = 6) radiochemical yield, respectively, with excellent radiochemical purity (>99%). Intravenous administration of both probes resulted in high tumor radioactivity accumulation (16.5 ± 2.8 and 8.6 ± 1.3 percentage injected dose per gram for apoA-I- and phospholipid-labeled rHDL, respectively) at 24 h after injection. Histologic analysis showed good colocalization of radioactivity with TAM-rich areas in tumor sections. Flow cytometry revealed high specificity of rHDL for TAMs, which had the highest uptake per cell (6.8-fold higher than tumor cells for both DiO@Zr-PL-HDL and DiO@Zr-AI-HDL) and accounted for 40.7% and 39.5% of the total cellular DiO@Zr-PL-HDL and DiO@Zr-AI-HDL in tumors, respectively. CONCLUSION:We have developed (89)Zr-labeled TAM imaging agents based on the natural nanoparticle rHDL. In an orthotopic mouse model of breast cancer, we have demonstrated their specificity for macrophages, a result that was corroborated by flow cytometry. Quantitative macrophage PET imaging with our (89)Zr-rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment.

Project description:Rationale: The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to CH2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates. Methods: We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO-nsspertuzumab) and two using variants of our chemoenzymatic protocol (DFO-sspertuzumab-EndoS and DFO-sspertuzumab-?Gal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human Fc?RI (huFc?RI), and mouse Fc?RI (muFc?RI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer. Results: MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO-sspertuzumab-EndoS possesses an attenuated binding affinity for huFc?RI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO-nsspertuzumab (4.1 ± 0.1 nM), and DFO-sspertuzumab-?Gal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89Zr-DFO-sspertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFc?RI. Conclusion: These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to Fc?RI.

Dataset Information

Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging.

Publications

Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging.

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