Jane K. Sosabowski

Prognostic and Therapeutic Impact of Argininosuccinate Synthetase 1 Control in Bladder Cancer as Monitored Longitudinally by PET Imaging

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [(18)F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response.

Radiolabelled peptides for oncological diagnosis

Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The 111In-labelled somatostatin analogue octreotide (OctreoScan™) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours.

Selection of Radiolabeled Gastrin Analogs for Peptide Receptor–Targeted Radionuclide Therapy

The gastrin/cholecystokinin-2 (CCK-2) receptor has been identified as a possible target for peptide receptor radionuclide imaging and therapy. Several radiolabeled peptides binding to this receptor have been explored in animal models and clinical trials but either low tumor uptake or high renal retention has been found. The aim of this study was to identify a peptide with improved tumor-to-kidney pharmacodynamics when compared with current candidates. Methods: A small peptide-chelator library of 34 compounds based on the C-terminal sequences of CCK-8 or minigastrin was constructed. The peptides were radiolabeled with 111In with high labeling efficiency (>90%), as determined by high-performance liquid chromatographic analysis. The labeled peptides were screened by assessing tumor and kidney uptake in pancreatic xenograft nude mouse models, including AR42J. An extensive biodistribution analysis was performed on the lead candidate from the library. Results: Minigastrin analogs containing a pentaglutamate sequence showed the highest tumor uptake but very high renal retention. CCK analogs showed the lowest tumor and renal uptake. Deletion of the pentaglutamate sequence in the gastrin analogs lowered the tumor uptake by a factor of 3 but decreased the kidney uptake by a factor of 20. Insertion of histidine residues in the sequence reduced kidney uptake by a further factor of almost 2-fold. In AR42J tumor-bearing mice, the peptide with the sequence DOTA-HHEAYGWMDF-NH2 (DOTA is tetraazacyclododecane tetraacetic acid) showed the highest tumor-to-kidney ratio of all peptides studied, with saturable uptake in target organs and low uptake by nontarget tissues other than the kidney. Conclusion: This peptide is a worthwhile candidate for clinical studies to determine whether it is suitable for use in peptide receptor–targeted radionuclide therapy.

Preclinical In Vivo Modeling of Cytokine Release Syndrome Induced by ErbB-Retargeted Human T Cells: Identifying a Window of Therapeutic Opportunity?

The ErbB network is dysregulated in many solid tumors. To exploit this, we have developed a chimeric Ag receptor (CAR) named T1E28z that targets several pathogenetically relevant ErbB dimers. T1E28z is coexpressed with a chimeric cytokine receptor named 4αβ (combination termed T4), enabling the selective expansion of engineered T cells using IL-4. Human T4+ T cells exhibit antitumor activity against several ErbB+ cancer types. However, ErbB receptors are also expressed in several healthy tissues, raising concerns about toxic potential. In this study, we have evaluated safety of T4 immunotherapy in vivo using a SCID beige mouse model. We show that the human T1E28z CAR efficiently recognizes mouse ErbB+ cells, rendering this species suitable to evaluate preclinical toxicity. Administration of T4+ T cells using the i.v. or intratumoral routes achieves partial tumor regression without clinical or histopathologic toxicity. In contrast, when delivered i.p., tumor reduction is accompanied by dose-dependent side effects. Toxicity mediated by T4+ T cells results from target recognition in both tumor and healthy tissues, leading to release of both human (IL-2/IFN-γ) and murine (IL-6) cytokines. In extreme cases, outcome is lethal. Both toxicity and IL-6 release can be ameliorated by prior macrophage depletion, consistent with clinical data that implicate IL-6 in this pathogenic event. These data demonstrate that CAR-induced cytokine release syndrome can be modeled in mice that express target Ag in an appropriate distribution. Furthermore, our findings argue that ErbB-retargeted T cells can achieve therapeutic benefit in the absence of unacceptable toxicity, providing that route of administration and dose are carefully optimized.

Magnetically Decorated Multiwalled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents

Carbon nanotubes (CNTs) have been proposed as one of the most promising nanomaterials to be used in biomedicine for their applications in drug/gene delivery as well as biomedical imaging. The present study developed radio-labeled iron oxide decorated multi-walled CNTs (MWNT) as dual magnetic resonance (MR) and single photon emission computed tomography (SPECT) imaging agents. Hybrids containing different amounts of iron oxide were synthesized by in situ generation. Physicochemical characterisations revealed the presence of superparamagnetic iron oxide nanoparticles (SPION) granted the magnetic properties of the hybrids. Further comprehensive examinations including high resolution transmission electron microscopy (HRTEM), fast Fourier transform simulations (FFT), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) assured the conformation of prepared SPION as γ-Fe2O3. High r2 relaxivities were obtained in both phantom and in vivo MRI compared to the clinically approved SPION Endorem®. The hybrids were successfully radio-labeled with technetium-99m through a functionalized bisphosphonate and enabled SPECT/CT imaging and γ-scintigraphy to quantitatively analyze the biodistribution in mice. No abnormality was found by histological examination and the presence of SPION and MWNT were identified by Perls stain and Neutral Red stain, respectively. TEM images of liver and spleen tissues showed the co-localization of SPION and MWNT within the same intracellular vesicles, indicating the in vivo stability of the hybrids after intravenous injection. The results demonstrated the capability of the present SPION-MWNT hybrids as dual MRI and SPECT contrast agents for in vivo use.

Conjugation of DOTA-like chelating agents to peptides and radiolabeling with trivalent metallic isotopes

Peptides can be labeled with various trivalent radiometals for imaging or targeted radionuclide-therapy applications. The peptide is first conjugated to a chelating agent that is able to form stable complexes with the radionuclide of interest. This conjugation step can be carried out as part of the solid-phase peptide synthesis, or it can be undertaken in the solution phase after synthesis and purification of the peptide. The latter route, described here, involves reacting a molar excess of the activated tri-tert-butyl ester-derivatized chelator with a designated free amino group of a peptide analog, in which all other reactive amines are protected, in the presence of a coupling agent. The conjugate molecule is then purified prior to deprotection and further purification by HPLC. The product can be radiolabeled by addition of a suitable metal salt, followed, if necessary, by removal of the unchelated metal. The entire process of conjugation, purification and radiolabeling should take approximately 12.5 h.

Targeting of CCK-2 Receptor–Expressing Tumors Using a Radiolabeled Divalent Gastrin Peptide

Gastrin/cholecystokinin subtype 2 receptors (CCK-2Rs) are overexpressed in several tumor types and are, thus, a potential target for peptide receptor radionuclide therapy (PRRT) of cancer. To improve the in vivo performance of CCK-2R binding peptides, we have previously synthesized and screened a series of divalent gastrin peptides for improved biochemical and biologic characteristics. In this study, we explore in more detail the most promising of these compounds and compare its performance with a previously described monomeric peptide. Methods: From six 111In-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA)–conjugated divalent gastrin peptides based on the C-terminal sequence of minigastrin, the maleimide-linked compound DOTA-GSC(succinimidopropionyl-EAYGWNleDF-NH2)-EAYGWNleDF-NH2 (MGD5) was selected. The in vitro stability, receptor binding, and internalization of 111In-MGD5 were studied and compared with those of monomer compound 111In-APH070. In vivo biodistribution and imaging using a SPECT/CT camera were also performed. Results: More than 90% of the labeled divalent peptide remained intact after 20 h of incubation in plasma. The inhibitory concentration of 50% of the divalent peptide was 1.0 versus 5.6 nM for the monomer, and the dissociation constant was 0.7 versus 2.9 nM. The rate of internalization of the divalent peptide was twice that of the monomer. Tumor uptake of the divalent peptide in vivo was about 6 times that of the monomer. The rate of washout of the divalent peptide from the tumor was lower than that of the monomer. Conclusion: Dimerization of the CCK-2R binding site results in an increase in binding affinity and an increase in tumor uptake both in vitro and in vivo. It is likely that these increases would result in improved tumor-targeting efficiency in patients with CCK-2R–positive tumors.

Tumor targeting and imaging with dual-peptide conjugated multifunctional liposomal nanoparticles.

Background The significant progress in nanotechnology provides a wide spectrum of nanosized material for various applications, including tumor targeting and molecular imaging. The aim of this study was to evaluate multifunctional liposomal nanoparticles for targeting approaches and detection of tumors using different imaging modalities. The concept of dual-targeting was tested in vitro and in vivo using liposomes derivatized with an arginine-glycine-aspartic acid (RGD) peptide binding to αvβ3 integrin receptors and a substance P peptide binding to neurokinin-1 receptors. Methods For liposome preparation, lipids, polyethylene glycol building blocks, DTPA-derivatized lipids for radiolabeling, lipid-based RGD and substance P building blocks and imaging labels were combined in defined molar ratios. Liposomes were characterized by photon correlation spectroscopy and zeta potential measurements, and in vitro binding properties were tested using fluorescence microscopy. Standardized protocols for radiolabeling were developed to perform biodistribution and micro-single photon emission computed tomography/computed tomography (SPECT/CT) studies in nude mice bearing glioblastoma and/or melanoma tumor xenografts. Additionally, an initial magnetic resonance imaging study was performed. Results Liposomes were radiolabeled with high radiochemical yields. Fluorescence microscopy showed specific cellular interactions with RGD-liposomes and substance P-liposomes. Biodistribution and micro-SPECT/CT imaging of 111In-labeled liposomal nanoparticles revealed low tumor uptake, but in a preliminary magnetic resonance imaging study with a single-targeted RGD-liposome, uptake in the tumor xenografts could be visualized. Conclusion The present study shows the potential of liposomes as multifunctional targeted vehicles for imaging of tumors combining radioactive, fluorescent, and magnetic resonance signaling. Specific in vitro tumor targeting by fluorescence microscopy and radioactivity was achieved. However, biodistribution studies in an animal tumor model revealed only moderate tumor uptake and no additive effect using a dual-targeting approach.

PET and SPECT imaging of a radiolabeled minigastrin analogue conjugated with DOTA, NOTA, and NODAGA and labeled with (64)Cu, (68)Ga, and (111)In.

Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.

The relationship between the diameter of chemically-functionalized multi-walled carbon nanotubes and their organ biodistribution profiles in vivo

Carbon nanotubes (CNTs) exhibit unique properties which have led to their applications in the biomedical field as novel delivery systems for diagnosis and therapy purposes. We have previously reported that the degree of functionalization of CNTs is a key factor determining their biological behaviour. The present study broadens the spectrum by investigating the impact of the diameter of CNTs using two series of multi-walled CNTs (MWNTs) with distinct differences in their diameters. Both MWNTs were doubly functionalized by 1,3-dipolar cycloaddition and amidation reactions, allowing the appended functional groups to be further conjugated with radionuclide chelating moieties and antibodies or antibody fragments. All constructs possessed comparable degree of functionalization and were characterized by thermogravimetric analysis, transmission electron microscopy, gel electrophoresis and surface plasmon resonance. The MWNT conjugates were radio-labelled with indium-111, which thereby enabled in vivo single photon emission computed tomography/computed tomography (SPECT/CT) imaging and organ biodistribution study using γ-scintigraphy. The narrow MWNTs (average diameter: 9.2 nm) demonstrated enhanced tissue affinity including non-reticular endothelial tissues compared to the wider MWNTs (average diameter: 39.5 nm). The results indicate that the higher aspect ratio of narrow MWNTs may be beneficial for their future biological applications due to higher tissue accumulation.