Dataset Information

ImmunoPET Imaging of CD146 Expression in Malignant Brain Tumors.

ABSTRACT: Recently, the overexpression of CD146 and its potential as a therapeutic target in high-grade gliomas, the most lethal type of brain cancer, was uncovered. In this study, we describe the generation of (89)Zr-Df-YY146, a novel (89)Zr-labeled monoclonal antibody (mAb) for the targeting and quantification of CD146 expression in a mouse model of glioblastoma, using noninvasive immunoPET imaging. YY146, a high affinity anti-CD146 mAb, was conjugated to deferoxamine (Df) for labeling with the long-lived positron emitter (89)Zr (t1/2: 78.4 h). In vitro assays, including flow cytometry, immunofluorescence microscopy, and Western blot, were performed with two glioblastoma cell lines, U87MG and U251, to determine their CD146 expression levels. Also, YY146 and Df-YY146's CD146-binding affinities were compared using flow cytometry. In vivo CD146-targeting of (89)Zr-Df-YY146 was evaluated by sequential PET imaging, in athymic nude mice bearing subcutaneously implanted U87MG or U251 tumors. CD146 blocking, ex vivo biodistribution, and histological studies were carried out to confirm (89)Zr-Df-YY146 specificity, as well as the accuracy of PET data. In vitro studies exposed elevated CD146 expression levels in U87MG cells, but negligible levels in U251 cells. Flow cytometry revealed no differences in affinity between YY146 and Df-YY146. (89)Zr labeling of Df-YY146 proceeded with excellent yield (?80%), radiochemical purity (>95%), and specific activity (?44 GBq/?mol). Longitudinal PET revealed prominent and persistent (89)Zr-Df-YY146 uptake in mice bearing U87MG tumors that peaked at 14.00 ± 3.28%ID/g (n = 4), 48 h post injection of the tracer. Conversely, uptake was significantly lower in CD146-negative U251 tumors (5.15 ± 0.99%ID/g, at 48 h p.i.; n = 4; P < 0.05). Uptake in U87MG tumors was effectively blocked in a competitive inhibition experiment, corroborating the CD146 specificity of (89)Zr-Df-YY146. Finally, ex vivo biodistribution validated the accuracy of PET data and histological examination successfully correlated tracer uptake with in situ CD146 expression. Prominent, persistent, and specific uptake of (89)Zr-Df-YY146 was observed in brain tumors, demonstrating the potential of this radiotracer for noninvasive PET imaging of CD146 expression. In a future clinical scenario, (89)Zr-Df-YY146 may serve as a tool to guide intervention and assess response to CD146-targeted therapies.

SUBMITTER: Hernandez R

PROVIDER: S-EPMC4935599 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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ImmunoPET Imaging of CD146 Expression in Malignant Brain Tumors.

Hernandez Reinier R Sun Haiyan H England Christopher G CG Valdovinos Hector F HF Barnhart Todd E TE Yang Yunan Y Cai Weibo W

Molecular pharmaceutics 20160610 7

Recently, the overexpression of CD146 and its potential as a therapeutic target in high-grade gliomas, the most lethal type of brain cancer, was uncovered. In this study, we describe the generation of (89)Zr-Df-YY146, a novel (89)Zr-labeled monoclonal antibody (mAb) for the targeting and quantification of CD146 expression in a mouse model of glioblastoma, using noninvasive immunoPET imaging. YY146, a high affinity anti-CD146 mAb, was conjugated to deferoxamine (Df) for labeling with the long-liv ...[more]

PMID: 27280694

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Project description:Overexpression of CD146 has been correlated with aggressiveness, recurrence rate, and poor overall survival in hepatocellular carcinoma (HCC) patients. In this study, we set out to develop a CD146-targeting probe for high-contrast noninvasive in vivo positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of HCCs. YY146, an anti-CD146 monoclonal antibody, was employed as a targeting molecule to which we conjugated the zwitterionic near-infrared fluorescence (NIRF) dye ZW800-1 and the chelator deferoxamine (Df). This enabled labeling of Df-YY146-ZW800 with (89)Zr and its subsequent detection using PET and NIRF imaging, all without compromising antibody binding properties. Two HCC cell lines expressing high (HepG2) and low (Huh7) levels of CD146 were employed to generate subcutaneous (s.c.) and orthotopic xenografts in athymic nude mice. Sequential PET and NIRF imaging performed after intravenous injection of (89)Zr-Df-YY146-ZW800 into tumor-bearing mice unveiled prominent and persistent uptake of the tracer in HepG2 tumors that peaked at 31.65 ± 7.15 percentage of injected dose per gram (%ID/g; n=4) 72 h post-injection. Owing to such marked accumulation, tumor delineation was successful by both PET and NIRF, which facilitated the fluorescence image-guided resection of orthotopic HepG2 tumors, despite the relatively high liver background. CD146-negative Huh7 and CD146-blocked HepG2 tumors exhibited significantly lower (89)Zr-Df-YY146-ZW800 accretion (6.1 ± 0.5 and 8.1 ± 1.0 %ID/g at 72 h p.i., respectively; n=4), demonstrating the CD146-specificity of the tracer in vivo. Ex vivo biodistribution and immunofluorescent staining corroborated the accuracy of the imaging data and correlated tracer uptake with in situ CD146 expression. Overall, (89)Zr-Df-YY146-ZW800 showed excellent properties as a PET/NIRF imaging agent, including high in vivo affinity and specificity for CD146-expressing HCC. CD146-targeted molecular imaging using dual-labeled YY146 has great potential for early detection, prognostication, and image-guided surgical resection of liver malignancies.

Project description:CD146 has been identified as an excellent biomarker for lung cancer as its overexpression in solid tumors has been linked to disease progression, invasion, and metastasis. Previously, our group described a positive correlation between 64Cu-labeled YY146 uptake and increased expression of CD146 in six human lung cancer cell lines using subcutaneous tumor models. In this study, we investigate a monoclonal antibody called YY146 for immunoPET imaging of CD146 in two intrapulmonary metastasis models of non-small cell lung cancer (NSCLC). The binding and immunoreactivity of the tracer were assessed by in vitro assays. Radiolabeling of YY146 with positron emitting Cu-64 (64Cu-NOTA-YY146) enabled PET imaging of intrapulmonary metastasis. Mice were intravenously injected with two million tumor cells, and CT imaging was used to verify the presence of lung metastases. 64Cu-NOTA-YY146 was injected into tumor-bearing mice, and animals were subjected to PET/CT imaging at 4, 24, and 48 h postinjection. Both the average and maximum lung PET signal intensities were quantified and compared between high and low CD146-expressing metastases. Further validation was accomplished through immunofluorescence imaging of resected tissues with CD31 and CD146. In flow cytometry, YY146 revealed strong binding to CD146 in H460 cells due to its high expression with minimal binding to CD146-low expressing H358 cells. Both YY146 and NOTA-YY146 showed similar binding, suggesting that NOTA conjugation did not elicit any negative effects on its binding affinity. Imaging of 64Cu-NOTA-YY146 in H460 tumor-bearing mice revealed rapid, persistent, and highly specific tracer accumulation. Uptake of 64Cu-NOTA-YY146 in the whole lung was calculated for H460 and H358 as 7.43 ± 0.38 and 3.95 ± 0.47% ID/g at 48 h postinjection (n = 4, p < 0.05), and the maximum lung signals were determined to be 13.85 ± 1.07 (H460) and 6.08 ± 0.73% ID/g (H358) at equivalent time points (n = 4, p < 0.05). To ensure the specificity of the tracer, a nonspecific antibody was injected into H460 tumor-bearing mice. Ex vivo biodistribution and immunofluorescence imaging validated the PET findings. In summary, 64Cu-NOTA-YY146 allowed for successful imaging of CD146-expressing intrapulmonary metastases of NSCLC in mice. This preliminary study provides evidence supporting the future clinical utilization of 64Cu-NOTA-YY146 for possible treatment monitoring of CD146-targeted therapy or improving patient stratification.

Project description:Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood-brain barrier (BBB) makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs), and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv) with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A "Trojan horse" method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT). Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs) are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs) and neural stem cells (NSCs) show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts), are making their way into glioma treatment as another type of cell-based therapy using the antibody to bind to the specific target(s). Finally, the current clinical trials are reviewed, showing the most recent progress of attractive approaches to deliver therapeutic antibodies across the BBB aiming at the specific antigen.

Project description:Overexpression of CD146 in solid tumors has been linked to disease progression, invasion, and metastasis. We describe the generation of a 64Cu-labeled CD146-specific antibody and its use for quantitative immunoPET imaging of CD146 expression in six lung cancer models.The anti-CD146 antibody (YY146) was conjugated to 1,4,7-triazacyclononane-triacetic acid (NOTA) and radiolabeled with 64Cu. CD146 expression was evaluated in six human lung cancer cell lines (A549, NCI-H358, NCI-H522, HCC4006, H23, and NCI-H460) by flow cytometry and quantitative western blot studies. The biodistribution and tumor uptake of 64Cu-NOTA-YY146 was assessed by sequential PET imaging in athymic nude mice bearing subcutaneous lung cancer xenografts. The correlation between CD146 expression and tumor uptake of 64Cu-NOTA-YY146 was evaluated by graphical software while ex vivo biodistribution and immunohistochemistry studies were performed to validate the accuracy of PET data and spatial expression of CD146.Flow cytometry and western blot studies showed similar findings with H460 and H23 cells showing high levels of expression of CD146. Small differences in CD146 expression levels were found among A549, H4006, H522, and H358 cells. Tumor uptake of 64Cu-NOTA-YY146 was highest in CD146-expressing H460 and H23 tumors, peaking at 20.1?±?2.86 and 11.6?±?2.34 %ID/g at 48 h after injection (n?=?4). Tumor uptake was lowest in the H522 model (4.1?±?0.98 %ID/g at 48 h after injection; n?=?4), while H4006, A549 and H358 exhibited similar uptake of 64Cu-NOTA-YY146. A positive correlation was found between tumor uptake of 64Cu-NOTA-YY146 (%ID/g) and relative CD146 expression (r 2?=?0.98, p?<?0.01). Ex vivo biodistribution confirmed the accuracy of the PET data.The strong correlation between tumor uptake of 64Cu-NOTA-YY146 and CD146 expression demonstrates the potential use of this radiotracer for imaging tumors that elicit varying levels of CD146. In the future, this tool may promote enhanced monitoring of therapeutic response and improved patient stratification.

Project description:PurposeTargeted delivery in vivo remains an immense roadblock for the translation of nanomaterials into the clinic. The greatest obstacle is the mononuclear phagocyte system (MPS), which sequesters foreign substances from general circulation and causes accumulation in organs such as the liver and spleen. The purpose of this study was to determine whether attaching an active targeting antibody, 5B1, to the surface of gold nanoparticles and using clodronate liposomes to deplete liver and splenic macrophages could help to minimize uptake by MPS organs, increase targeted delivery to CA19.9-positive pancreatic tumors, and enhance pancreatic tumor delineation.ProceduresTo produce the antibody-gold nanoparticle conjugate (Ab-AuNP), the Ab was conjugated to p-isothiocyanatobenzyl-desferrioxamine (p-SCN-DFO) and subsequently conjugated to NHS-activated gold nanoparticles. The Ab-AuNP was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modified Lindmo assay was performed to assess binding affinity and internalization potential in vitro. The Ab-AuNP was radiolabeled with 89Zr and injected into CA19.9-positive BxPc-3 pancreatic orthotopic tumor-bearing mice pretreated with or without clodronate liposomes for PET imaging and biodistribution studies. Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis was used to confirm delivery of gold nanoparticles to BxPc-3 pancreatic subcutaneous xenografts.ResultsMice pretreated with clodronate liposomes in an orthotopic setting demonstrated decreased liver uptake at early time points (12.2 ± 2.3 % ID/g vs. 22.8 ± 3.8 % ID/g at 24 h) and increased tumor uptake at 120 h (13.8 ± 8.0 % ID/g vs. 6.0 ± 1.2 % ID/g). This allowed for delineation of orthotopic pancreatic xenografts in significantly more mice treated with clodronate (6/6) than in mice not treated with clodronate (2/6) or mice injected with gold nanoparticles labeled with a nonspecific antibody (0/5).ConclusionsThe combination of clodronate liposomes and an active targeting antibody on the surface of gold nanoparticles allowed for PET/CT imaging of subcutaneous and orthotopic pancreatic xenografts in mice.

Dataset Information

ImmunoPET Imaging of CD146 Expression in Malignant Brain Tumors.

Publications

ImmunoPET Imaging of CD146 Expression in Malignant Brain Tumors.

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