Haiming Luo

Intrinsically Germanium-69-Labeled Iron Oxide Nanoparticles: Synthesis and In-Vivo Dual-Modality PET/MR Imaging

Intrinsically germanium-69-labeled super-paramagnetic iron oxide nanoparticles are synthesized via a newly developed, fast and highly specific chelator-free approach. The biodistribution pattern and the feasibility of (69) Ge-SPION@PEG for in vivo dual-modality positron emission tomography/magnetic resonance (PET/MR) imaging and lymph-node mapping are investigated, which represents the first example of the successful utilization of a (69) Ge-based agent for PET/MR imaging.

In Vivo Integrity and Biological Fate of Chelator-Free Zirconium-89-Labeled Mesoporous Silica Nanoparticles.

Traditional chelator-based radio-labeled nanoparticles and positron emission tomography (PET) imaging are playing vital roles in the field of nano-oncology. However, their long-term in vivo integrity and potential mismatch of the biodistribution patterns between nanoparticles and radio-isotopes are two major concerns for this approach. Here, we present a chelator-free zirconium-89 (89Zr, t1/2 = 78.4 h) labeling of mesoporous silica nanoparticle (MSN) with significantly enhanced in vivo long-term (>20 days) stability. Successful radio-labeling and in vivo stability are demonstrated to be highly dependent on both the concentration and location of deprotonated silanol groups (−Si–O–) from two types of silica nanoparticles investigated. This work reports 89Zr-labeled MSN with a detailed labeling mechanism investigation and long-term stability study. With its attractive radio-stability and the simplicity of chelator-free radio-labeling, 89Zr-MSN offers a novel, simple, and accurate way for studying the in vivo long-term fate and PET image-guided drug delivery of MSN in the near future.

HaloTag technology: a versatile platform for biomedical applications.

Exploration of protein function and interaction is critical for discovering links among genomics, proteomics, and disease state; yet, the immense complexity of proteomics found in biological systems currently limits our investigational capacity. Although affinity and autofluorescent tags are widely employed for protein analysis, these methods have been met with limited success because they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows protein function and interaction to be comprehensively analyzed using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review, we examine all current applications of the HaloTag technology platform for biomedical applications, such as the study of protein isolation and purification, protein function, protein–protein and protein–DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed along with potential future applications.

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

Significance Given the success of combination therapies for the treatment of cancer, the use of bispecific antibodies targeting multiple cancerous molecular pathways is an attractive strategy to enhance the efficacy of current therapeutic paradigms. However, parallel development of companion diagnostic tools is essential for patient identification, stratification, and the early assessment of treatment efficacies. Herein, we describe the generation of a bispecific construct for noninvasive PET imaging of glioblastoma via bioorthogonal click chemistry. The excellent tumor-homing properties displayed by our bispecific probe, which features two antibody fragments simultaneously targeting epidermal growth factor receptor and CD105, demonstrated that our approach is a simple and effective method to generate multispecific targeting agents for noninvasive molecular imaging. 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 64Cu-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 64Cu-NOTA-Bs-F(ab)2. 64Cu-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.

PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment

The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use 64Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Methods: Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for 64Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using 64Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. Results: NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of 64Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. Conclusion: 64Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. 64Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA.

Chelator-Free Labeling of Layered Double Hydroxide Nanoparticles for in Vivo PET Imaging

Layered double hydroxide (LDH) nanomaterial has emerged as a novel delivery agent for biomedical applications due to its unique structure and properties. However, in vivo positron emission tomography (PET) imaging with LDH nanoparticles has not been achieved. The aim of this study is to explore chelator-free labeling of LDH nanoparticles with radioisotopes for in vivo PET imaging. Bivalent cation 64Cu2+ and trivalent cation 44Sc3+ were found to readily label LDH nanoparticles with excellent labeling efficiency and stability, whereas tetravalent cation 89Zr4+ could not label LDH since it does not fit into the LDH crystal structure. PET imaging shows that prominent tumor uptake was achieved in 4T1 breast cancer with 64Cu-LDH-BSA via passive targeting alone (7.7 ± 0.1%ID/g at 16 h post-injection; n = 3). These results support that LDH is a versatile platform that can be labeled with various bivalent and trivalent radiometals without comprising the native properties, highly desirable for PET image-guided drug delivery.

Design and Applications of Bispecific Heterodimers: Molecular Imaging and beyond

Ligand-based molecular imaging probes have been designed with high affinity and specificity for monitoring biological process and responses. Single-target recognition by traditional probes can limit their applicability for disease detection and therapy because synergistic action between disease mediators and different receptors is often involved in disease progression. Consequently, probes that can recognize multiple targets should demonstrate higher targeting efficacy and specificity than their monospecific peers. This concept has been validated by multiple bispecific heterodimer-based imaging probes that have demonstrated promising results in several animal models. This review summarizes the design strategies for bispecific peptide- and antibody-based heterodimers and their applications in molecular targeting and imaging. The design and application of bispecific heterodimer-conjugated nanomaterials are also discussed.

PET Imaging of VEGFR-2 Expression in Lung Cancer with 64Cu-Labeled Ramucirumab

Lung cancer accounts for 17% of cancer-related deaths worldwide, and most patients present with locally advanced or metastatic disease. Novel PET imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be used for detecting VEGFR-2–positive malignancies and subsequent monitoring of therapeutic response to VEGFR-2–targeted therapies. Here, we report the synthesis and characterization of an antibody-based imaging agent for PET imaging of VEGFR-2 expression in vivo. Methods: Ramucirumab (named RamAb), a fully humanized IgG1 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with 64Cu. Flow cytometry analysis and microscopy studies were performed to compare the VEGFR-2 binding affinity of RamAb and NOTA-RamAb. PET imaging and biodistribution studies were performed in nude mice bearing HCC4006 and A549 xenograft tumors. Ex vivo histopathology was performed to elucidate the expression patterns of VEGFR-2 in different tissues and organs to validate in vivo results. Results: Flow cytometry examination revealed the specific binding capacity of fluorescein isothiocyanate-RamAb to VEGFR-2, and no difference in VEGFR-2 binding affinity was seen between RamAb and NOTA-RamAb. After being labeled with 64Cu, PET imaging revealed specific and prominent uptake of 64Cu-NOTA-RamAb in VEGFR-2–positive HCC4006 tumors (9.4 ± 0.5 percentage injected dose per gram at 48 h after injection; n = 4) and significantly lower uptake in VEGFR-2–negative A549 tumors (4.3 ± 0.2 percentage injected dose per gram at 48 h after injection; n = 3). Blocking experiments revealed significantly lower uptake in HCC4006 tumors, along with histology analysis, further confirming the VEGFR-2 specificity of 64Cu-NOTA-RamAb. Conclusion: This study provides initial evidence that 64Cu-NOTA-RamAb can function as a PET imaging agent for visualizing VEGFR-2 expression in vivo, which may also find potential applications in monitoring the treatment response of VEGFR-2–targeted cancer therapy.

Dual-targeting of tissue factor and CD105 for preclinical PET imaging of pancreatic cancer

Purpose: Pancreatic adenocarcinoma is a highly aggressive cancer, currently treated with limited success and dismal outcomes. New diagnostic and treatment strategies offer the potential to reduce cancer mortality. Developing highly specific noninvasive imaging probes for pancreatic cancer is essential to improving diagnostic accuracy and monitoring therapeutic intervention. Experimental Design: A bispecific heterodimer was synthesized by conjugating an anti-tissue factor (TF) Fab with an anti-CD105 Fab, via the bio-orthogonal “click” reaction between tetrazine (Tz) and trans-cyclooctene (TCO). The heterodimer was labeled with 64Cu for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic tumors. Results: PET imaging of BXPC-3 (TF/CD105+/+) xenograft tumors with 64Cu-labeled heterodimer displayed significantly enhanced tumor uptake (28.8 ± 3.2 %ID/g; n = 4; SD) at 30 hours postinjection, as compared with each of their monospecific Fab tracers (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; n = 3; SD). In addition, the activity–concentration ratio allowed for effective tumor visualization (tumor/muscle ratio 75.2 ± 9.4 at 30 hours postinjection.; n = 4; SD). Furthermore, 64Cu-NOTA-heterodimer enabled sensitive detection of orthotopic pancreatic tumor lesions with an uptake of 17.1 ± 4.9 %ID/g at 30 hours postinjection and tumor/muscle ratio of 72.3 ± 46.7. Conclusions: This study demonstrates that dual targeting of TF and CD105 provided synergistic improvements in binding affinity and tumor localization of the heterodimer. Dual-targeted imaging agents of pancreatic and other cancers may assist in diagnosing pancreatic malignancies as well as reliable monitoring of therapeutic response. Clin Cancer Res; 22(15); 3821–30. ©2016 AACR.

PET of c-Met in Cancer with 64Cu-Labeled Hepatocyte Growth Factor

The hepatocyte growth factor (HGF) and its receptor, c-Met, are actively involved in tumor progression and metastasis and are closely associated with a poor prognostic outcome for cancer patients. Thus, the development of PET agents that can assess c-Met expression would be extremely useful for diagnosing cancer and subsequently monitoring response to c-Met–targeted therapies. Here, we report the characterization of recombinant human HGF (rh-HGF) as a PET tracer for detection of c-Met expression in vivo. Methods: rh-HGF was expressed in human embryonic kidney 293 cells and purified by nickel-nitrilotriacetic acid affinity chromatography. The concentrated rh-HGF was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid and labeled with 64Cu. c-Met binding evaluation by flow cytometry was performed on both U87MG and MDA-MB-231 cell lines, which have a high level and a low level, respectively, of c-Met. PET imaging and biodistribution studies were performed on nude mice bearing U87MG and MDA-MB-231 xenografted tumors. Results: The rh-HGF expression yield was 150–200 μg of protein per 5 × 106 cells after a 48-h transfection, with purity of approximately 85%–90%. Flow cytometry examination confirmed that rh-HGF had a strong and specific capacity to bind to c-Met. After 64Cu labeling, PET imaging revealed specific and prominent uptake of 64Cu-NOTA-rh-HGF in c-Met–positive U87MG tumors (percentage injected dose per gram, 6.8 ± 1.8 at 9 h after injection) and significantly lower uptake in c-Met–negative MDA-MB-231 tumors (percentage injected dose per gram, 1.8 ± 0.6 at 9 h after injection). The fact that sonication-denatured rh-HGF had significantly lower uptake in U87MG tumors, along with histology analysis, confirmed the c-Met specificity of 64Cu-NOTA-rh-HGF. Conclusion: This study provided initial evidence that 64Cu-NOTA-rh-HGF visualizes c-Met expression in vivo, an application that may prove useful for c-Met–targeted cancer therapy.