CD146-Targeted Multimodal Image-Guided Photoimmunotherapy of Melanoma.
ABSTRACT: For melanoma resistant to molecularly targeted therapy and immunotherapy, new treatment strategies are urgently needed. A molecularly targeted theranostic pair may thus be of importance, where the diagnostic probe facilitates patient stratification and the therapeutic companion treats the selected cases. For this purpose, flow cytometry is used to assess the CD146 level in melanoma cells. Based on YY146, a CD146-specific monoclonal antibody, an imaging probe 89Zr-Df-YY146 is synthesized and its diagnostic performance is evaluated by positron emission tomography (PET) imaging. Furthermore, a photoimmunotherapy (PIT) agent IR700-YY146 is developed and the therapeutic effect of IR700-YY146 PIT is assessed comprehensively. CD146 is highly expressed in A375 and SK-MEL-5 cells. 89Zr-Df-YY146 PET readily detects CD146-positive A375 melanomas. Tumor accumulation of 89Zr-Df-YY146 peaks at 72 h with an uptake value of 26.48 ± 3.28%ID g-1, whereas the highest uptake of the nonspecific 89Zr-Df-IgG is 4.80 ± 1.75%ID g-1. More importantly, IR700-YY146 PIT effectively inhibits the growth of A375 tumors, owing to production of reactive oxygen species, decreased glucose metabolism, and reduced expression of CD146. To conclude, 89Zr-Df-YY146 and IR700-YY146 are a promising theranostic pair with the former revealing CD146 expression in melanoma as a PET probe and the latter specifically treating CD146-positive melanoma as an effective PIT agent.
Project description: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.
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: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:With advancement in antibody engineering, the development and characterization of new cancer-specific molecular targets are in the forefront of this PET-antibody combination "revolution". Overexpression of CD146 in different types of tumors, including breast tumor, has been associated with tumor progression and poor prognosis. Non-invasive detection of CD146 with a monoclonal antibody may provide a noninvasive diagnostic tool with high specificity and accountability.<h4>Methods</h4>Herein, we have developed a CD146-specific monoclonal antibody (YY146), radiolabeled it with <sup>52</sup>Mn and <sup>89</sup>Zr and identified its capability in acting as a non-invasive imaging agent that specific targets CD146 in different murine breast cancer models. CD146 expression was first screened in different breast tumor cell lines through Western Blot and confirmed its binding ability to YY146 using Flow Cytometry. Serial immunoPET images were carried out after intravenous administration of <sup>52</sup>Mn or <sup>89</sup>Zr labeled YY146. In addition, we also performed in vivo fluorescence imaging in animals injected with YY146 conjugated with Cy5.5.<h4>Results</h4>Western Blot results show that MDA-MB-435 cell line had greater levels of CD146 expression when compared to the other cell lines investigated. Flow cytometry confirmed binding ability of YY146. PET images revealed well correlated uptake between tumor uptake and CD146 expression levels, confirmed by biodistribution studies and fluorescence imaging.<h4>Conclusion</h4>PET imaging, for up to 7 days, of mice bearing three different breast tumors were carried out and revealed radiotracer uptake in tumors that strongly (r<sup>2</sup> = 0.98, P < 0.01), correlated with CD146 expression levels, as confirmed by in vitro and ex vivo studies.
Project description:Given the highly heterogeneous character of brain malignancies and the associated implication for its proper diagnosis and treatment, finding biomarkers that better characterize this disease from a molecular standpoint is imperative. In this study, we evaluated CD146 as a potential molecular target for diagnosis and targeted therapy of glioblastoma multiforme (GBM), the most common and lethal brain malignancy. YY146, an anti-CD146 monoclonal antibody, was generated and radiolabeled for noninvasive positron-emission tomography (PET) imaging of orthotopic GBM models. (64)Cu-labeled YY146 preferentially accumulated in the tumors of mice bearing U87MG xenografts, which allowed the acquisition of high-contrast PET images of small tumor nodules (∼ 2 mm). Additionally, we found that tumor uptake correlated with the levels of CD146 expression in a highly specific manner. We also explored the potential therapeutic effects of YY146 on the cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) properties of U87MG cells, demonstrating that YY146 can mitigate those aggressive phenotypes. Using YY146 as the primary antibody, we performed histological studies of World Health Organization (WHO) grades I through IV primary gliomas. The positive correlation found between CD146-positive staining and high tumor grade (χ(2) = 9.028; P = 0.029) concurred with the GBM data available in The Cancer Genome Atlas (TCGA) and validated the clinical value of YY146. In addition, we demonstrate that YY146 can be used to detect CD146 in various cancer cell lines and human resected tumor tissues of multiple other tumor types (gastric, ovarian, liver, and lung), indicating a broad applicability of YY146 in solid tumors.
Project description:Mounting evidence suggests that the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a central role in tumor progression. The goal of this study was to develop an 89Zr-labeled, antibody-based positron emission tomography (PET) tracer for quantitative imaging of the uPA/uPAR system. An anti-uPA monoclonal antibody (ATN-291) was conjugated with a deferoxamine (Df) derivative and subsequently labeled with 89Zr. Flow cytometry, microscopy studies, and competitive binding assays were conducted to validate the binding specificity of Df-ATN-291 against uPA. PET imaging with 89Zr-Df-ATN-291 was carried out in different tumors with distinct expression levels of uPA. Biodistribution, histology examination, and Western blotting were performed to correlate tumor uptake with uPA or uPAR expression. ATN-291 retained uPA binding affinity and specificity after Df conjugation. 89Zr-labeling of ATN-291 was achieved in good radiochemical yield and high specific activity. Serial PET imaging demonstrated that, in most tumors studied (except uPA- LNCaP), the uptake of 89Zr-Df-ATN-291 was higher compared to major organs at 120 h post-injection, providing excellent tumor contrast. The tumor-to-muscle ratio of 89Zr-Df-ATN-291 in U87MG was as high as 45.2 ± 9.0 at 120 h p.i. In vivo uPA specificity of 89Zr-Df-ATN-291 was confirmed by successful pharmacological blocking of tumor uptake with ATN-291 in U87MG tumors. Although the detailed mechanisms behind in vivo 89Zr-Df-ATN-291 tumor uptake remained to be further elucidated, quantitative PET imaging with 89Zr-Df-ATN-291 in tumors can facilitate oncologists to adopt more relevant cancer treatment planning.
Project description:Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89Zr-Df-ALT-836 for TF in vivo. 89Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. Flow cytometry and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5±6.0%ID/g at 48h post-injection; n=3), which was significantly abrogated (2.3±0.5%ID/g at 48h post-injection; n=3) when mice were pre-injected with a blocking dose (50mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89Zr-Df-ALT-836. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients.
Project description:Pembrolizumab is a humanized monoclonal antibody targeting programmed cell death protein 1 (PD-1) found on T and pro-B cells. Pembrolizumab prevents PD-1 ligation by both PD-L1 and PD-L2, preventing the immune dysregulation that otherwise occurs when T-cells encounter cells expressing these ligands. Clinically, PD-1 blockade elicits potent antitumor immune responses, and antibodies blocking PD-1 ligation, including pembrolizumab, have recently received Food and Drug Administration approval for the treatment of advanced melanoma, renal cell cancer, and non-small cell lung cancer. METHODS:In this study, we evaluated the pharmacokinetics, biodistribution, and dosimetry of pembrolizumab in vivo, accomplished through radiolabeling with the positron emitter 89Zr. PET imaging was used to evaluate the whole-body distribution of 89Zr-deferoxamine (Df)-pembrolizumab in two rodent models (mice and rats). Data obtained from PET scans and biodistribution studies were extrapolated to humans to estimate the dosimetry of the tracer. As a proof of concept, the biodistribution of 89Zr-Df-pembrolizumab was further investigated in a humanized murine model. RESULTS:The tracer remained stable in blood circulation throughout the study and accumulated the greatest in liver and spleen tissues. Both mice and rats showed similar biodistribution and pharmacokinetics of 89Zr-Df-pembrolizumab. In the humanized mouse model, T-cell infiltration into the salivary and lacrimal glands could be successfully visualized. CONCLUSION:These data will augment our understanding of the pharmacokinetics and biodistribution of radiolabeled pembrolizumab in vivo, while providing detailed dosimetry data that may lead to better dosing strategies in the future. These findings further demonstrate the utility of noninvasive in vivo PET imaging to dynamically track T-cell checkpoint receptor expression and localization in a humanized mouse model.
Project description:Anti-epidermal growth factor receptor (EGFR) antibody therapy is used in EGFR expressing cancers including lung, colon, head and neck, and bladder cancers, however results have been modest. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate which is activated by NIR light. NIR-PIT is in clinical trials in patients with recurrent head and neck cancers using cetuximab-IR700 as the conjugate. However, its use has otherwise been restricted to mouse models. This is an effort to explore larger animal models with NIR-PIT. We describe the use of a recombinant canine anti-EGFR monoclonal antibody (mAb), can225IgG, conjugated to the photo-absorber, IR700DX, in three EGFR expressing canine transitional cell carcinoma (TCC) cell lines as a prelude to possible canine clinical studies. Can225-IR700 conjugate showed specific binding and cell-specific killing after NIR-PIT on EGFR expressing cells in vitro. In the in vivo study, can225-IR700 conjugate demonstrated accumulation of the fluorescent conjugate with high tumor-to-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 µg of can225-IR700 i.v. only; (3) NIR light exposure only; (4) 100 µg of can225-IR700 i.v., NIR light exposure. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups (p < 0.001), and significantly prolonged survival was achieved (p < 0.001 vs. other groups) in the treatment groups. In conclusion, NIR-PIT with can225-IR700 is a promising treatment for canine EGFR-expressing cancers, including invasive transitional cell carcinoma in pet dogs, that could provide a pathway to translation to humans.