Sukhen C. Ghosh

Dual-Labeling Strategies for Nuclear and Fluorescence Molecular Imaging: A Review and Analysis

Molecular imaging is used for the detection of biochemical processes through the development of target-specific contrast agents. Separately, modalities such as nuclear and near-infrared fluorescence (NIRF) imaging have been shown to non-invasively monitor disease. More recently, merging of these modalities has shown promise owing to their comparable detection sensitivity and benefited from the development of dual-labeled imaging agents. Dual-labeled agents hold promise for whole-body and intraoperative imaging and could bridge the gap between surgical planning and image-guided resection with a single, molecularly targeted agent. In this review, we summarized the literature for dual-labeled antibodies and peptides that have been developed and have highlighted key considerations for incorporating NIRF dyes into nuclear labeling strategies. We also summarized our findings on several commercially available NIRF dyes and offer perspectives for developing a toolkit to select the optimal NIRF dye and radiometal combination for multimodality imaging.

A peptide probe for targeted brown adipose tissue imaging

The presence of brown adipose tissue (BAT) responsible for thermogenic energy dissipation has been revealed in adult humans and has high clinical importance. Due to limitations of current methods for BAT detection, analyzing the abundance and localization of BAT in the body has remained challenging. Here, we screen a combinatorial peptide library in mice and characterize a peptide (with the sequence CPATAERPC) that selectively binds to the vascular endothelium of BAT, but not of intraperitoneal white adipose tissue (WAT). We show that in addition to BAT, this peptide probe also recognizes the vasculature of BAT-like depots of subcutaneous WAT. Our results indicate that the CPATAERPC peptide localizes to BAT even in the absence of sympathetic nervous system stimulation. Finally, we demonstrate that this probe can be used to identify BAT depots in mice by whole body near-infrared (NIR) fluorescence imaging.

Multimodal Chelation Platform for Near-Infrared Fluorescence/Nuclear Imaging

Dual-labeled compounds containing nuclear and near-infrared fluorescence contrast have the potential to molecularly guide surgical resection of cancer by extending whole-body diagnostic imaging findings into the surgical suite. To simplify the dual labeling process for antibody-based agents, we designed a multimodality chelation (MMC) scaffold which combined a radiometal chelating agent and fluorescent dye into a single moiety. Three dye-derivatized MMC compounds were synthesized and radiolabeled. The IRDye 800CW conjugate, 4, had favorable optical properties and showed rapid clearance in vivo. Using 4, an epithelial cell adhesion molecule (EpCAM) targeting MMC-immunoconjugate was prepared and dual-labeled with (64)Cu. In vitro binding activity was confirmed after MMC conjugation. Multimodal imaging studies showed higher tumor accumulation of (64)Cu-7 compared to nontargeted (64)Cu-4 in a prostate cancer model. Further evaluation in different EpCAM-expressing cell lines is warranted as well as application of the MMC dual labeling approach with other monoclonal antibodies.

Targeting Pili in Enterococcal Pathogenesis

ABSTRACT Passive protection, the administration of antibodies to prevent infection, has garnered significant interest in recent years as a potential prophylactic countermeasure to decrease the prevalence of hospital-acquired infections. Pili, polymerized protein structures covalently anchored to the peptidoglycan wall of many Gram-positive pathogens, are ideal targets for antibody intervention, given their importance in establishing infection and their accessibility to antibody interactions. In this work, we demonstrated that a monoclonal antibody to the major component of Enterococcus faecalis pili, EbpC, labels polymerized pilus structures, diminishes biofilm formation, and significantly prevents the establishment of a rat endocarditis infection. The effectiveness of this anti-EbpC monoclonal provides strong evidence in support of its potential as a preventative. In addition, after radiolabeling, this monoclonal identified the site of enterococcal infection, providing a rare example of molecularly specific imaging of an established bacterial infection and demonstrating the versatility of this agent for use in future diagnostic and therapeutic applications.

Comparison of DOTA and NODAGA as chelators for 64Cu-labeled immunoconjugates

INTRODUCTION Bifunctional chelators have been shown to impact the biodistribution of monoclonal antibody (mAb)-based imaging agents. Recently, radiolabeled 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-peptide complexes have demonstrated improved in vivo stability and performance compared to their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) counterparts. Here, we investigated if similar utility could be achieved with mAbs and compared (64)Cu-labeled DOTA and NODAGA-immunoconjugates for the detection of epithelial cell adhesion molecule (EpCAM) in a prostate cancer model. METHODS DOTA and NODAGA-immunoconjugates of an EpCAM targeting mAb (mAb7) were synthesized and radiolabeled with (64)Cu (DOTA: 40°C for 1hr; NODAGA: 25°C for 1hr). The average number of chelators per mAb was quantified by isotopic dilution, and the biological activity of the immunoconjugates was evaluated by flow cytometry and ELISA. Radioligand assays were performed to compare cellular uptake and determine the dissociation constant (Kd) and maximum number of binding sites (Bmax) for the immunoconjugates using DsRed-transfected PC3-cells. A PC3-DsRed xenograft tumor model was established in nude mice and used to perform biodistribution studies to compare organ uptake and pharmacokinetics. RESULTS (64)Cu-DOTA-mAb7 and (64)Cu-NODAGA-mAb7 were prepared with chelator/protein ratios of 2-3 and obtained in comparable radiochemical yields ranging from 59 to 71%. Similar immunoreactivity was observed with both agents, and mock labeling studies indicated that incubation at room temperature or 40°C did not affect potency. (64)Cu-NODAGA-mAb7 demonstrated higher in vitro cellular uptake while (64)Cu-DOTA-mAb7 had higher Kd and Bmax values. From the biodistribution data, we found similar tumor uptake (13.44±1.21%ID/g and 13.24±4.86%ID/g for (64)Cu-DOTA-mAb7 and (64)Cu-NODAGA-mAb7, respectively) for both agents at 24hr, although normal prostate tissue was significantly lower for (64)Cu-NODAGA-mAb7. (64)Cu-NODAGA-mAb7 also had less accumulation in the liver, suggesting excellent retention of the chelation complex in vivo. This was further confirmed by the higher blood activity of (64)Cu-NODAGA-mAb7, which corresponds to increased bioavailability afforded by the enhanced in vivo stability of the agent. Although tumor/muscle ratios were comparable, tumor/prostate ratios were >2-fold and 1.5-fold higher for (64)Cu-NODAGA-mAb7 at 24 and 48hr, respectively, and suggest better ability to discriminate tumor tissue with (64)Cu-NODAGA-mAb7 in our prostate cancer model. CONCLUSIONS To the best of our knowledge, this study represents the first comparison of (64)Cu-labeled DOTA and NODAGA immunoconjugates in vivo. Our results show favorable in vivo performance for (64)Cu-NODAGA-mAb7 which builds upon previous data on our hybrid mAb7 imaging agent by increasing the detection sensitivity for metastatic prostate tumors, as well as for other types of cancer that express EpCAM.

LGR5-Targeted Antibody–Drug Conjugate Eradicates Gastrointestinal Tumors and Prevents Recurrence

Gastrointestinal cancer is one of the leading causes of cancer-related mortality in men and women worldwide. The adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein–coupled receptor 5) is highly expressed in a significant fraction of gastrointestinal tumors of the colon, liver, pancreas, and stomach, relative to normal tissues. LGR5 is located on the cell surface and undergoes rapid, constitutive internalization independent of ligand. Furthermore, LGR5-high cancer cells have been shown to exhibit the properties of tumor-initiating cells or cancer stem cells (CSC). On the basis of these attributes, we generated two LGR5-targeting antibody–drug conjugates (ADC) by tethering the tubulin-inhibiting cytotoxic drug monomethyl auristatin E to a highly specific anti-LGR5 mAb via a protease cleavable or noncleavable chemical linker and compared them in receptor binding, cell internalization, and cytotoxic efficacy in cancer cells. Here, we show that both ADCs bind LGR5 with high specificity and equivalent nanomolar affinity and rapidly internalize to the lysosomes of LGR5-expressing gastrointestinal cancer cells. The anti-LGR5 ADCs effectively induced cytotoxicity in LGR5-high gastrointestinal cancer cells, but not in LGR5-negative or -knockdown cancer cell lines. Overall, we demonstrate that the cleavable ADC exhibited higher potency in vitro and was able to eradicate tumors and prevent recurrence in a xenograft model of colon cancer. These findings provide preclinical evidence for the potential of LGR5-targeting ADCs as effective new therapeutics for the treatment and eradication of gastrointestinal tumors and CSCs with high LGR5 expression. Mol Cancer Ther; 15(7); 1580–90. ©2016 AACR.

Advances in the Development of Multimodal Imaging Agents for Nuclear/Near-infrared Fluorescence Imaging.

Multimodal imaging agents were first introduced a decade ago and consist of a targeting moiety that is dual-labeled with radioactive and fluorescent contrast. These compounds allow whole-body and intraoperative imaging to be performed through administration of a single agent and provide complementary diagnostic information that can be used to guide tumor resection. Since their initial evaluation, interest in dual-labeled agents has continued to grow and their design has subsequently evolved alongside the development of novel chelating agents, improved fluorophores, and highly selective coupling techniques for bioconjugate formation. In this review, will discuss how changes in the labeling components and schemes for multimodal agent development have impacted imaging performance and will focus on antibody- and peptide-based agents as models for dual labeling. We will also describe the growing role of modular dual labeling strategies as well as direct labeling methods using radiohalogens.

Molecular inhibition of prostaglandin E2 with GW627368X: Therapeutic potential and preclinical safety assessment in mouse sarcoma model

Prostaglandin E2, the major COX-2 product, acts via 4 functionally distinct prostanoid receptors, EP(1–4). PGE-2, through its receptors, feeds back to positively increase COX-2 expression augmenting its own synthesis thereby driving angiogenesis, while suppressing apoptosis and innate immunity. In addition to the well characterized PGE2/EP4/cAMP/PKA/CREB, EP4 activation increases GSK3 phosphorylation via PI3K and Akt consequently reducing β-catenin phosphorylation. EP4 induces angiogenesis by enhancing VEGF production via ERK activation. These effects of EP4 are asserted either directly or via EGFR transactivation depending on the type of cancer. In view of the safety concerns regarding long term use of COX-2 inhibitors and to find more effective alternatives, we evaluated the potential of EP4 prostanoid receptor as a target for treating cancer progression using a highly selective EP4 antagonist, 4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl)-N-(phenylsulfonyl)-benzeneacetamide. Oral administration of GW627368X showed significant tumor regression characterized by tumor reduction and induction of apoptosis. Reduction in prostaglandin E2 synthesis also led to reduced level of VEGF in plasma. Regulation of multiple pathways downstream of EP4 was evident by down regulation of COX-2, p-Akt, p-MAPK and p-EGFR. Considering wide distribution of the EP4 prostanoid receptor in major organs and the array of physiological processes it contributes to, the safety profile of the drug was analyzed. No major organ toxicity, immunosupression, behavioral change or change in blood parameters attributable to the drug was observed. The results assert the significance of EP4 prostanoid receptor as a therapeutic target as well as the safety of EP4 blockade by GW627368X.

Synthesis of a Fluorescently Labeled 68Ga-DOTA-TOC Analog for Somatostatin Receptor Targeting

Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog, 68Ga-DOTA-TOC, to produce the dual-labeled analog, 68Ga-MMC(IRDye 800CW)-TOC, with high yield and specific activity. In vitro pharmacological assays demonstrated retention of receptor-targeting properties for the dual-labeled compound with robust internalization that was somatostatin receptor (SSTR) 2-mediated. Biodistribution studies in mice identified the kidneys as the primary excretion route for 68Ga-MMC(IRDye 800CW)-TOC, along with clearance via the reticuloendothelial system. Higher uptake was observed in most tissues compared to 68Ga-DOTA-TOC but decreased as a function of time. The combination of excellent specificity for SSTR2-expressing cells and suitable biodistribution indicate potential application of 68Ga-MMC(IRDye 800CW)-TOC for intraoperative detection of SSTR2-expressing tumors.

Response to di-functionalized hyaluronic acid with orthogonal chemistry grafting at independent modification sites in rodent models of neural differentiation and spinal cord injury

Hyaluronic acid (HA) with one reactive moiety grafted to the backbone is a commonly used matrix in tissue engineering. The addition of a second orthogonal moiety to the backbone allows for greater control in bioactive signal tethering and gelation. In this study, thiol and azide functional groups were grafted to the HA backbone at separate modification sites. NMR, FT-IR, colorimetric assay, and radio-TLC activity were used to confirm and quantify thiol and azide grafting to the HA backbone. Various ratios of di-functional HA (dif HA) and methacrylate HA (mHA) were used to encapsulate mouse embryonic stem cells in order to examine the neural differentiation of the cells. Greater neural maturation was observed in hydrogels containing a higher percentage of dif HA compared to mHA over a six day neural differentiation time course. This formulation was then tested in a contusion spinal cord injury model for biological effect and was found to reduce the ED1+ area in the spinal cord compared to control and allow for host axon extension into the matrix filled lesion area. These results indicate that dif HA is supportive of neural differentiation and can reduce inflammation without additional bioactive signal tethering. dif HA is a promising matrix base for the central nervous system, which should be further developed.