Evaluation of the biological activities of the IL-15 superagonist complex, ALT-803, following intravenous versus subcutaneous administration in murine models.

ALT-803 is a fusion protein complex consisting of an interleukin (IL)-15 superagonist and a dimeric IL-15 receptor alpha sushi domain IgG1 Fc fusion protein. When administered to mice, ALT-803 is capable of inducing natural killer (NK) and CD8+ T cell proliferation and activation, and effectively promoting potent anti-tumor responses. Currently, ALT-803 is in clinical trials for treatment of various solid tumors and hematological malignancies. In the initial phase of these clinical studies, intravenous (iv) injection was used according to the route used in pre-clinical efficacy studies. In order to evaluate the possible advantage of subcutaneous (sc) injection versus iv injection, this study compared the biological activity of the two treatment regimens of ALT-803 in pre-clinical in vivo models. The pharmacokinetics, immune stimulation, and anti-tumor efficacy of iv and sc injection routes of ALT-803 in C57BL/6 mice were compared. The half-life of ALT-803 was 7.5 h for iv versus 7.7 h for sc with the maximal detected serum concentration of ALT-803 to be 3926 ng/ml at 0.5 h time-point following iv injection versus 495 ng/ml at 16 h post sc injection. Biodistribution studies indicated that sc ALT-803, similarly to iv ALT-803 as previously reported, has a greater tissue distribution and longer residence time in lymphoid tissues compared to recombinant IL-15. Notably, ALT-803 when administered either iv or sc induced comparable proliferation and activation of CD8+ T and NK cells and resulted in similar reductions of tumor burden. A toxicity study of mice receiving multiple injections of ALT-803 for 4 weeks by iv or sc routes revealed equivalent immune-related changes. The gradual absorbance into the blood stream and lower maximal blood levels of ALT-803 in sc-injected mice, along with similar anti-tumor efficacy support the administration of ALT-803 by sc injection in patients with various malignancies and infectious diseases.

89Zr-labeled nivolumab for imaging of T-cell infiltration in a humanized murine model of lung cancer.

PURPOSE: Nivolumab is a human monoclonal antibody specific for programmed cell death-1 (PD-1), a negative regulator of T-cell activation and response. Acting as an immune checkpoint inhibitor, nivolumab binds to PD-1 expressed on the surface of many immune cells and prevents ligation by its natural ligands. Nivolumab is only effective in a subset of patients, and there is limited evidence supporting its use for diagnostic, monitoring, or stratification purposes. METHODS: 89Zr-Df-nivolumab was synthesized to map the biodistribution of PD-1-expressing tumor infiltrating T-cells in vivo using a humanized murine model of lung cancer. The tracer was developed by radiolabeling the antibody with the positron emitter zirconium-89 (89Zr). Imaging results were validated by ex vivo biodistribution studies, and PD-1 expression was validated by immunohistochemistry. Data obtained from PET imaging were used to determine human dosimetry estimations. RESULTS: The tracer showed elevated binding to stimulated PD-1 expressing T-cells in vitro and in vivo. PET imaging of 89Zr-Df-nivolumab allowed for clear delineation of subcutaneous tumors through targeting of localized activated T-cells expressing PD-1 in the tumors and salivary glands of humanized A549 tumor-bearing mice. In addition to tumor uptake, salivary and lacrimal gland infiltration of T-cells was noticeably visible and confirmed via histological analysis. CONCLUSIONS: These data support our claim that PD-1-targeted agents allow for tumor imaging in vivo, which may assist in the design and development of new immunotherapies. In the future, noninvasive imaging of immunotherapy biomarkers may assist in disease diagnostics, disease monitoring, and patient stratification.

ImmunoPET imaging of CD38 in murine lymphoma models using 89Zr-labeled daratumumab.

PURPOSE: CD38 is considered a potential biomarker for multiple myeloma (MM) and has shown a strong link with chronic lymphocytic leukemia due to high and uniform expression on plasma cells. In vivo evaluation of CD38 expression may provide useful information about lesion detection and prognosis of treatment in MM. In this study, immunoPET imaging with 89Zr-labeled daratumumab was used for differentiation of CD38 expression in murine lymphoma models to provide a potential non-invasive method for monitoring CD38 in the clinic. METHODS: Daratumumab was radiolabeled with 89Zr (t1/2 = 78.4 h) via conjugation with desferrioxamine (Df). After Western blot (WB) was used to screen CD38 expression in five lymphoma cell lines, flow cytometry and cellular binding assays were performed to test the binding ability of labeled or conjugated daratumumab with CD38 in vitro. PET imaging and biodistribution studies were performed to evaluate CD38 expression after injection of 89Zr-Df-daratumumab. 89Zr-Df-IgG was also evaluated as a non-specific control group in the Ramos model. Finally, CD38 expression in tumor tissues was verified by histological analysis. RESULTS: Using WB screening, the Ramos cell line was found to express the highest level of CD38 while the HBL-1 cell line had the lowest expression. Df-conjugated and 89Zr-labeled daratumumab displayed similar high binding affinities with Ramos cells. PET imaging of 89Zr-Df-daratumumab showed a high tumor uptake of up to 26.6 ± 8.0 %ID/g for Ramos at 120 h post-injection, and only up to 6.6 ± 2.9 %ID/g for HBL-1 (n = 4). Additionally, 89Zr-Df-IgG demonstrated a low tumor uptake in the Ramos model (only 4.3 ± 0.8 %ID/g at 120 h post-injection). Ex vivo biodistribution studies showed similar trends with imaging results. Immunofluorescence staining of tumor tissues verified higher CD38 expression of Ramos than that of HBL-1. CONCLUSIONS: The role of 89Zr-Df-daratumumab was investigated for evaluating CD38 expression in lymphoma models non-invasively and was found to be to a promising imaging agent of CD38-positive hematological diseases such as MM in future clinical applications.

Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry.

Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal "click" ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105(+/+)) tumors with (64)Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of (64)Cu-NOTA-Bs-F(ab)2. (64)Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1-labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions.

Targeting CD146 with a 64Cu-labeled antibody enables in vivo immunoPET imaging of high-grade gliomas.

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.

Renal-Clearable PEGylated Porphyrin Nanoparticles for Image-guided Photodynamic Cancer Therapy.

Noninvasive dynamic positron emission tomography (PET) imaging was used to investigate the balance between renal clearance and tumor uptake behaviors of polyethylene glycol (PEG)-modified porphyrin nanoparticles (TCPP-PEG) with various molecular weights. TCPP-PEG10K nanoparticles with clearance behavior would be a good candidate for PET image-guided photodynamic therapy.

Lymphoma: current status of clinical and preclinical imaging with radiolabeled antibodies.

Lymphoma is a complex disease that arises from cells of the immune system with an intricate pathology. While lymphoma may be classified as Hodgkin or non-Hodgkin, each type of tumor is genetically and phenotypically different and highly invasive tissue biopsies are the only method to investigate these differences. Noninvasive imaging strategies, such as immunoPET, can provide a vital insight into disease staging, monitoring treatment response in patients, and dose planning in radioimmunotherapy. ImmunoPET imaging with radiolabeled antibody-based tracers may also assist physicians in optimizing treatment strategies and enhancing patient stratification. Currently, there are two common biomarkers for molecular imaging of lymphoma, CD20 and CD30, both of which have been considered for investigation in preclinical imaging studies. In this review, we examine the current status of both preclinical and clinical imaging of lymphoma using radiolabeled antibodies. Additionally, we briefly investigate the role of radiolabeled antibodies in lymphoma therapy. As radiolabeled antibodies play critical roles in both imaging and therapy of lymphoma, the development of novel antibodies and the discovery of new biomarkers may greatly affect lymphoma imaging and therapy in the future.

Radiolabeled pertuzumab for imaging of human epidermal growth factor receptor 2 expression in ovarian cancer.

PURPOSE: Human epidermal growth factor receptor 2 (HER2) is over-expressed in over 30% of ovarian cancer cases, playing an essential role in tumorigenesis and metastasis. Non-invasive imaging of HER2 is of great interest for physicians as a mean to better detect and monitor the progression of ovarian cancer. In this study, HER2 was assessed as a biomarker for ovarian cancer imaging using 64Cu-labeled pertuzumab for immunoPET imaging. METHODS: HER2 expression and binding were examined in three ovarian cancer cell lines (SKOV3, OVCAR3, Caov3) using in vitro techniques, including western blot and saturation binding assays. PET imaging and biodistribution studies in subcutaneous models of ovarian cancer were performed for non-invasive in vivo evaluation of HER2 expression. Additionally, orthotopic models were employed to further validate the imaging capability of 64Cu-NOTA-pertuzumab. RESULTS: HER2 expression was highest in SKOV3 cells, while OVCAR3 and Caov3 displayed lower HER2 expression. 64Cu-NOTA-pertuzumab showed high specificity for HER2 (Ka = 3.1 ± 0.6 nM) in SKOV3. In subcutaneous tumors, PET imaging revealed tumor uptake of 41.8 ± 3.8, 10.5 ± 3.9, and 12.1 ± 2.3%ID/g at 48 h post-injection for SKOV3, OVCAR3, and Caov3, respectively (n = 3). In orthotopic models, PET imaging with 64Cu-NOTA-pertuzumab allowed for rapid and clear delineation of both primary and small peritoneal metastases in HER2-expressing ovarian cancer. CONCLUSIONS: 64Cu-NOTA-pertuzumab is an effective PET tracer for the non-invasive imaging of HER2 expression in vivo, rendering it a potential tracer for treatment monitoring and improved patient stratification.

ImmunoPET Imaging of CD146 in Murine Models of Intrapulmonary Metastasis of Non-Small Cell Lung Cancer.

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.

CD38 as a PET Imaging Target in Lung Cancer.

Daratumumab (Darzalex, Janssen Biotech) is a clinically approved antibody targeting CD38 for the treatment of multiple myeloma. However, CD38 is also expressed by other cancer cell types, including lung cancer, where its expression or absence may offer prognostic value. We therefore developed a PET tracer based upon daratumumab for tracking CD38 expression, utilizing murine models of non-small cell lung cancer to verify its specificity. Daratumumab was prepared for radiolabeling with 89Zr (t1/2 = 78.4 h) through conjugation with desferrioxamine (Df). Western blot, flow cytometry, and saturation binding assays were utilized to characterize CD38 expression and binding of daratumumab to three non-small cell lung cancer cell lines: A549, H460, and H358. Murine xenograft models of the cell lines were also generated for further in vivo studies. Longitudinal PET imaging was performed following injection of 89Zr-Df-daratumumab out to 120 h postinjection, and nonspecific uptake was also evaluated through the injection of a radiolabeled control IgG antibody in A549 mice, 89Zr-Df-IgG. Ex vivo biodistribution and histological analyses were also performed after the terminal imaging time point at 120 h postinjection. Through cellular studies, A549 cells were found to express higher levels of CD38 than the H460 or H358 cell lines. PET imaging and ex vivo biodistribution studies verified in vitro trends, with A549 tumor uptake peaking at 8.1 ± 1.2%ID/g at 120 h postinjection according to PET analysis, and H460 and H358 at lower levels at the same time point (6.7 ± 0.7%ID/g and 5.1 ± 0.4%ID/g, respectively; n = 3 or 4). Injection of a nonspecific radiolabeled IgG into A549 tumor-bearing mice also demonstrated lower tracer uptake of 4.4 ± 1.3%ID/g at 120 h. Immunofluorescent staining of tumor tissues showed higher staining levels present in A549 tissues over H460 and H358. Thus, 89Zr-Df-daratumumab is able to image CD38-expressing tissues in vivo using PET, as verified through the exploration of non-small cell lung cancer models in this study. This agent therefore holds potential to image CD38 in other malignancies and aid in patient stratification and elucidation of the biodistribution of CD38.

ImmunoPET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer with a Dual-Labeled Bispecific Antibody Fragment.

Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab' antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide (64Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF+/+) or PANC-1 (CD105/TF-/-) tumor xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of 64Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4%ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0%ID/g; n = 4; p < 0.01) and ALT-836 (7.6 ± 3.7%ID/g; n = 4; p < 0.01) at 24 h postinjection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by 4-fold with TF blocking and 2-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1%ID/g; n = 4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated that the dual targeting with 64Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like 64Cu-NOTA-heterodimer-ZW800, may increase the overall tumor accumulation in comparison to single-targeted homodimers, leading to improved imaging of cancer and other related diseases.

ImmunoPET Imaging of CTLA-4 Expression in Mouse Models of Non-small Cell Lung Cancer.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is expressed on the surface of activated T cells and some tumor cells, and is the target of the clinically approved monoclonal antibody ipilimumab. In this study, we investigate specific binding of radiolabeled ipilimumab to CTLA-4 expressed by human non-small cell lung cancer cells in vivo using positron emission tomography (PET). Ipilimumab was radiolabeled with 64Cu (t1/2 = 12.7 h) through the use of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to formulate 64Cu-DOTA-ipilimumab. CTLA-4 expression in three non-small cell lung cancer (NSCLC) cell lines (A549, H460, and H358) was verified and quantified by Western blot and enzyme-linked immunosorbent assays (ELISA). A receptor binding assay was utilized to monitor the binding and internalization of 64Cu-DOTA-ipilimumab in the NSCLC cell lines. Next, the biodistribution of 64Cu-DOTA-ipilimumab was mapped by longitudinal PET imaging up to 48 h after injection. Ex vivo biodistribution and histological studies were employed to verify PET results. By in vitro analysis, CTLA-4 was found to be expressed on all three NSCLC cell lines with A549 and H358 showing the highest and lowest level of expression, respectively. PET imaging and quantification verified these findings as the tracer accumulated highest in the A549 tumor model (9.80 ± 0.22%ID/g at 48 h after injection; n = 4), followed by H460 and H358 tumors with uptakes of 9.37 ± 0.26%ID/g and 7.43 ± 0.05%ID/g, respectively (n = 4). The specificity of the tracer was verified by injecting excess ipilimumab in A549 tumor-bearing mice, which decreased tracer uptake to 6.90 ± 0.51%ID/g at 48 after injection (n = 4). Ex vivo analysis following the last imaging session also corroborated these findings. 64Cu-DOTA-ipilimumab showed enhanced and persistent accumulation in CTLA-4-expressing tissues, which will enable researchers further insight into CTLA-4 targeted therapies in the future.

Chelator-Free Radiolabeling of Nanographene: Breaking the Stereotype of Chelation.

Macrocyclic chelators have been widely employed in the realm of nanoparticle-based positron emission tomography (PET) imaging, whereas its accuracy remains questionable. Here, we found that 64 Cu can be intrinsically labeled onto nanographene based on interactions between Cu and the π electrons of graphene without the need of chelator conjugation, providing a promising alternative radiolabeling approach that maintains the native in vivo pharmacokinetics of the nanoparticles. Due to abundant π bonds, reduced graphene oxide (RGO) exhibited significantly higher labeling efficiency in comparison with graphene oxide (GO) and exhibited excellent radiostability in vivo. More importantly, nonspecific attachment of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) on nanographene was observed, which revealed that chelator-mediated nanoparticle-based PET imaging has its inherent drawbacks and can possibly lead to erroneous imaging results in vivo.

NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.

The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a nonideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system.

ImmunoPET for assessing the differential uptake of a CD146-specific monoclonal antibody in lung cancer.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Molecular Imaging of Pancreatic Cancer with Antibodies.

Development of novel imaging probes for cancer diagnostics remains critical for early detection of disease, yet most imaging agents are hindered by suboptimal tumor accumulation. To overcome these limitations, researchers have adapted antibodies for imaging purposes. As cancerous malignancies express atypical patterns of cell surface proteins in comparison to noncancerous tissues, novel antibody-based imaging agents can be constructed to target individual cancer cells or surrounding vasculature. Using molecular imaging techniques, these agents may be utilized for detection of malignancies and monitoring of therapeutic response. Currently, there are several imaging modalities commonly employed for molecular imaging. These imaging modalities include positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance (MR) imaging, optical imaging (fluorescence and bioluminescence), and photoacoustic (PA) imaging. While antibody-based imaging agents may be employed for a broad range of diseases, this review focuses on the molecular imaging of pancreatic cancer, as there are limited resources for imaging and treatment of pancreatic malignancies. Additionally, pancreatic cancer remains the most lethal cancer with an overall 5-year survival rate of approximately 7%, despite significant advances in the imaging and treatment of many other cancers. In this review, we discuss recent advances in molecular imaging of pancreatic cancer using antibody-based imaging agents. This task is accomplished by summarizing the current progress in each type of molecular imaging modality described above. Also, several considerations for designing and synthesizing novel antibody-based imaging agents are discussed. Lastly, the future directions of antibody-based imaging agents are discussed, emphasizing the potential applications for personalized medicine.

ImmunoPET Imaging of CD146 Expression in Malignant Brain Tumors.

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/µmol). 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.

ImmunoPET Imaging of Insulin-Like Growth Factor 1 Receptor in a Subcutaneous Mouse Model of Pancreatic Cancer.

The role of insulin-like growth factor-1 receptor (IGF-1R) in cancer tumorigenesis was established decades ago, yet there are limited studies evaluating the imaging and therapeutic properties of anti-IGF-1R antibodies. Noninvasive imaging of IGF-1R may allow for optimized patient stratification and monitoring of therapeutic response in patients. Herein, this study reports the development of a Zirconium-89 ((89)Zr)-labeled anti-IGF-1R antibody ((89)Zr-Df-1A2G11) for PET imaging of pancreatic cancer. Successful chelation and radiolabeling of the antibody resulted in a highly stable construct that could be used for imaging IGF-1R expressing tumors in vivo. Western blot and flow cytometry studies showed that MIA PaCa-2, BxPC-3, and AsPC-1 pancreatic cancer cell lines expressed high, moderate, and low levels of IGF-1R, respectively. These three pancreatic cancer cell lines were subcutaneously implanted into mice. By employing the PET imaging technique, the tumor accumulation of (89)Zr-Df-1A2G11 was found to be dependent on the level of IGF-1R expression. Tumor accumulation of (89)Zr-Df-1A2G11 was 8.24 ± 0.51, 5.80 ± 0.54, and 4.30 ± 0.42 percentage of the injected dose (%ID/g) in MIA PaCa-2, BxPC-3, and AsPC-1-derived tumor models at 120 h postinjection, respectively (n = 4). Biodistribution studies and ex vivo immunohistochemistry confirmed these findings. In addition, (89)Zr-labeled nonspecific human IgG ((89)Zr-Df-IgG) displayed minimal uptake in IGF-1R positive MIA PaCa-2 tumor xenografts (3.63 ± 0.95%ID/g at 120 h postinjection; n = 4), demonstrating that (89)Zr-Df-1A2G11 accumulation was highly specific. This study provides initial evidence that our (89)Zr-labeled IGF-1R-targeted antibody may be employed for imaging a wide range of malignancies. Antibodies may be tracked in vivo for several days to weeks with (89)Zr, which may enhance image contrast due to decreased background signal. In addition, the principles outlined in this study can be employed for identifying patients that may benefit from anti-IGF-1R therapy.