Julie L. Sutcliffe

Arginine Deiminase as a Novel Therapy for Prostate Cancer Induces Autophagy and Caspase-Independent Apoptosis

Arginine deprivation as an anticancer therapy has historically been met with limited success. The development of pegylated arginine deiminase (ADI-PEG20) has renewed interest in arginine deprivation for the treatment of some cancers. The efficacy of ADI-PEG20 is directly correlated with argininosuccinate synthetase (ASS) deficiency. CWR22Rv1 prostate cancer cells do not express ASS, the rate-limiting enzyme in arginine synthesis, and are susceptible to ADI-PEG20 in vitro. Interestingly, apoptosis by 0.3 microg/mL ADI-PEG20 occurs 96 hours posttreatment and is caspase independent. The effect of ADI-PEG20 in vivo reveals reduced tumor activity by micropositron emission tomography as well as reduced tumor growth as a monotherapy and in combination with docetaxel against CWR22Rv1 mouse xenografts. In addition, we show autophagy is induced by single amino acid depletion by ADI-PEG20. Here, autophagy is an early event that is detected within 1 to 4 hours of 0.3 microg/mL ADI-PEG20 treatment and is an initial protective response to ADI-PEG20 in CWR22Rv1 cells. Significantly, the inhibition of autophagy by chloroquine and Beclin1 siRNA knockdown enhances and accelerates ADI-PEG20-induced cell death. PC3 cells, which express reduced ASS, also undergo autophagy and are responsive to autophagy inhibition and ADI-PEG20 treatment. In contrast, LNCaP cells highly express ASS and are therefore resistant to both ADI-PEG20 and autophagic inhibition. These data point to an interrelationship among ASS deficiency, autophagy, and cell death by ADI-PEG20. Finally, a tissue microarray of 88 prostate tumor samples lacked expression of ASS, indicating ADI-PEG20 is a potential novel therapy for the treatment of prostate cancer

Use of a Peptide Derived from Foot-and-Mouth Disease Virus for the Noninvasive Imaging of Human Cancer: Generation and Evaluation of 4-[18F]Fluorobenzoyl A20FMDV2 for In vivo Imaging of Integrin αvβ6 Expression with Positron Emission Tomography

Expression of the epithelial-specific integrin alphavbeta6 is low or undetectable in most adult tissues but may be increased during wound healing and inflammation and is up-regulated dramatically by many different carcinomas, making alphavbeta6 a promising target for the in vivo detection of cancer using noninvasive imaging. In addition, alphavbeta6 is recognized as promoting invasion and correlates with aggressive behavior of human cancers and thus agents that recognize alphavbeta6 specifically in vivo will be an essential tool for the future management of alphavbeta6-positive cancers. Recently, we identified the peptide NAVPNLRGDLQVLAQKVART (A20FMDV2), derived from foot-and-mouth disease virus, as a potent inhibitor of alphavbeta6. Using flow cytometry and ELISA, we show that this peptide is highly selective, inhibiting alphavbeta6-ligand binding with a IC50 of 3 nmol/L, an activity 1,000-fold more selective for alphavbeta6 than for other RGD-directed integrins (alphavbeta3, alphavbeta5, and alpha5beta1). A20FMDV2 was radiolabeled on solid-phase using 4-[18F]fluorobenzoic acid, injected into mice bearing both alphavbeta6-negative and alphavbeta6-positive (DX3puro/DX3purobeta6 cell lines) xenografts and imaged using a small animal positron emission tomography (PET) scanner. Rapid uptake (<30 min) and selective retention (>5 h) of radioactivity in the alphavbeta6-positive versus the alphavbeta6-negative tumor, together with fast renal elimination of nonspecifically bound activity, resulted in specific imaging of the alphavbeta6-positive neoplasm. These data suggest that PET imaging of alphavbeta6-positive tumors is feasible and will provide an important new tool for early detection and improved management of many types of cancers.

Targeted In vivo Imaging of Integrin αvβ6 with an improved Radiotracer and Its Relevance in a Pancreatic Tumor Model

The cell surface receptor alpha(v)beta(6) is epithelial specific, and its expression is tightly regulated; it is low or undetectable in adult tissues but has been shown to be increased in many different cancers, including pancreatic, cervical, lung, and colon cancers. Studies have described alpha(v)beta(6) as a prognostic biomarker linked to poor survival. We have recently shown the feasibility of imaging alpha(v)beta(6) in vivo by positron emission tomography (PET) using the peptide [(18)F]FBA-A20FMDV2. Here, we describe improved alpha(v)beta(6) imaging agents and test their efficacy in a mouse model with endogenous alpha(v)beta(6) expression. The modified compounds maintained high affinity for alpha(v)beta(6) and >1,000-fold selectivity over related integrins (by ELISA) and showed significantly improved alpha(v)beta(6)-dependent binding in cell-based assays (>60% binding versus <10% for [(18)F]FBA-A20FMDV2). In vivo studies using either a melanoma cell line (transduced alpha(v)beta(6) expression) or the BxPC-3 human pancreatic carcinoma cell line (endogenous alpha(v)beta(6) expression) revealed that the modified compounds showed significantly improved tumor retention. This, along with good clearance of nonspecifically bound activity, particularly for the new radiotracer [(18)F]FBA-PEG(28)-A20FMDV2, resulted in improved PET imaging. Tumor/pancreas and tumor/blood biodistribution ratios of >23:1 and >47:1, respectively, were achieved at 4 hours. Significantly, [(18)F]FBA-PEG(28)-A20FMDV2 was superior to 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG) in imaging the BxPC-3 tumors. Pancreatic ductal adenocarcinoma is highly metastatic and current preoperative evaluation of resectability using noninvasive imaging has limited success, with most patients having metastases at time of surgery. The fact that these tumors express alpha(v)beta(6) suggests that this probe has significant potential for the in vivo detection of this malignancy, thus having important implications for patient care and therapy.

Structure-Function Analysis of Arg-Gly-Asp Helix Motifs in αvβ6 Integrin Ligands

Data relating to the structural basis of ligand recognition by integrins are limited. Here we describe the physical requirements for high affinity binding of ligands to αvβ6. By combining a series of structural analyses with functional testing, we show that 20-mer peptide ligands, derived from high affinity ligands of αvβ6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix. This arrangement allows two conserved Leu/Ile residues at Asp+1 and Asp+4 to be presented on the outside face of the helix enabling a potential hydrophobic interaction with the αvβ6 integrin, in addition to the Arg-Gly-Asp interaction. The extent of the helix determines peptide affinity for αvβ6 and potency as an αvβ6 antagonist. A major role of this C-terminal helix is likely to be the correct positioning of the Asp+1 and Asp+4 residues. These data suggest an explanation for several biological functions of αvβ6 and provide a structural platform for design of αvβ6 antagonists.

Preclinical evaluation of carcinoembryonic cell adhesion molecule (CEACAM) 6 as potential therapy target for pancreatic adenocarcinoma.

Despite the availability of new targeted therapies, ductal pancreatic adenocarcinoma continues to carry a poor prognosis. Carcinoembryonic antigen‐related cell adhesion molecule (CEACAM)6 has been reported as a potential biomarker and therapy target for this malignancy. We have evaluated CEACAM6 as a potential therapy target, using an antibody–drug conjugate (ADC). Expression of CEACAM6 in pancreatic adenocarcinomas was determined using immunohistochemistry on tissue microarrays. The expression pattern in granulocytes and granulocytic precursors was measured by flow cytometry. Murine xenograft and non‐human primate models served to evaluate efficacy and safety, respectively. Robust expression of CEACAM6 was found in > 90% of invasive pancreatic adenocarcinomas as well as in intraepithelial neoplastic lesions. In the granulocytic lineage, CEACAM6 was expressed at all stages of granulocytic maturation except for the early lineage‐committed precursor cell. The anti‐CEACAM6 ADC showed efficacy against established CEACAM6‐expressing tumours. In non‐human primates, antigen‐dependent toxicity of the ADC consisted of dose‐dependent and reversible depletion of granulocytes and their precursors. This was associated with preferential and rapid localization of the antibody in bone marrow, as determined by sequential in vivo PET imaging of the radiolabelled anti‐CEACAM6. Localization of the radiolabelled tracer could be attenuated by predosing with unlabelled antibody confirming specific accumulation in this compartment. Based on the expression pattern in normal and malignant pancreatic tissues, efficacy against established tumours and limited and reversible bone marrow toxicity, we propose that CEACAM6 should be considered for an ADC‐based therapy approach against pancreatic adenocarcinomas and possibly other CEACAM6‐positive neoplasms. Copyright

High-throughput in vivo screening of targeted molecular imaging agents

The rapid development and translation of targeted molecular imaging agents from bench to bedside is currently a slow process, with a clear bottleneck between the discovery of new compounds and the development of an appropriate molecular imaging agent. The ability to identify promising new molecular imaging agents, as well as failures, much earlier in the development process using high-throughput screening techniques could save significant time and money. This work combines the advantages of combinatorial chemistry, site-specific solid-phase radiolabeling, and in vivo imaging for the rapid screening of molecular imaging agents. A one-bead-one-compound library was prepared and evaluated in vitro, leading to the identification of 42 promising lead peptides. Over 11 consecutive days, these peptides, along with a control peptide, were successfully radiolabeled with 4-[18F]fluorobenzoic acid and evaluated in vivo using microPET. Four peptides were radiolabeled per day, followed by simultaneous injection of each individual peptide into 2 animals. As a result, 4 promising new molecular imaging agents were identified that otherwise would not have been selected based solely on in vitro data. This study is the first example of the practical application of a high-throughput screening approach using microPET imaging of [18F]-labeled peptides for the rapid in vivo identification of potential new molecular imaging agents.

Radiolabeling rhesus monkey CD34+ hematopoietic and mesenchymal stem cells with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for microPET imaging.

Noninvasive positron emission tomography (PET) provides a potential method for in vivo tracking of radiolabeled cells. The goal of this study was to assess the potential toxicity of 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM) on rhesus monkey CD34+ hematopoietic and mesenchymal stem cells in vitro in preparation for developing imaging protocols posttransplantation. CD34+ hematopoietic cells were radiolabeled with 0 to 40 μCi/mL 64Cu-PTSM and viability and colony formation were assessed. Rhesus monkey mesenchymal stem cells (rhMSCs) were placed in culture postradiolabeling for assessments of growth and differentiation toward adipogenic, osteogenic, and chondrogenic lineages. The results indicated that CD34+ cells radiolabeled with 20 μCi/mL and rhMSCs radiolabeled with 10 μCi/mL 64Cu-PTSM did not result in adverse effects on growth or differentiation. Nonradioactive copper was also evaluated and showed that the presence of copper was not harmful to the cells. CD34+ cells and rhMSCs radiolabeled with the optimized concentrations of 20 and 10 μCi/mL, respectively, were also assessed using the microPET scanner. Studies showed that a minimum of 2.50 × 104 CD34+ cells (1.1 pCi/cell) and 6.25 × 103 rhMSCs (4.4 pCi/cell) could be detected. These studies indicate that CD34+ hematopoietic cells and rhMSCs can be safely radiolabeled with 64Cu-PTSM without adverse cellular effects.

Preoperative evaluation of pancreatic adenocarcinoma

The preoperative evaluation of resectability for pancreatic cancer fails to identify up to 25% of patients who are unfortunately found to be unresectable at surgical exploration. Inoperative findings in this circumstance is usually due to either small volume metastatic disease or regional tumor invasion. While advances in computed tomography (CT) technology has increased accuracy of local tumor extent, occult metastatic disease remains a common problem. Although 2-[(18)F]fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) has been demonstrated to be useful in the staging of many malignancies (e.g. esophageal cancer, recurrent colorectal cancer, lung cancer), it has not been found to significantly increase the accuracy of determining resectability preoperatively in pancreatic cancer, especially with regard to detection of small volume metastatic disease. There are a variety of pancreatic cancer-specific antigens which are being developed as a method for targeted molecular imaging; we provide preliminary data targeting the integrin alpha(v)beta(6) to demonstrate the potential feasibility of this approach. Further developments may allow the accurate determination of patients with resectable pancreatic cancer, and more importantly, those with unresectable disease that may forego unnecessary surgery, the associated morbidity, and the subsequent delay of appropriate therapy.

Copper-Free Click for PET: Rapid 1,3-Dipolar Cycloadditions with a Fluorine-18 Cyclooctyne

The strain-promoted click 1,3-dipolar cycloaddition reactions involving azides and cyclooctynes for the synthesis of triazoles offer the advantage of being able to be performed in biological settings via copper-free chemistries. While strained reagents conjugated to optical dyes and radiometal conjugates have been reported, cyclooctyne reagents labeled with fluorine-18 ((18)F) and radiochemically evaluated in a copper-free click reaction have yet to be explored. This report describes the conversion of a bifunctional azadibenzocyclooctyne (ADIBO) amine to the (18)F-labeled cyclooctyne 4, the subsequent fast copper-free 1,3-dipolar cycloaddition reaction with alkyl azides at 37 °C (>70% radiochemical conversion in 30 min), and biological evaluations (serum stability of >95% at 2 h). These findings demonstrate the excellent reactivity of the (18)F-labeled cyclooctyne 4 with readily available azides that will allow future work focusing on rapid copper-free in vitro and in vivo click chemistries for PET imaging using (18)F-labeled cyclooctyne derivatives of ADIBO.

Dynamic imaging of arginine-rich heart-targeted vehicles in a mouse model

Efficacious delivery of drugs and genes to the heart is an important goal. Here, a radiolabeled peptide-targeted liposome was engineered to bind to the heart, and the biodistribution and pharmacokinetics were determined by dynamic positron emission tomography in the FVB mouse. Efficient targeting occurred only with an exposed ligand and a dense concentration of peptide (6000 peptides/particles). Liposomes targeted with CRPPR or other arginine-rich peptides with an exposed guanidine moiety bound within 100 s after intravenous injection and remained stably bound. With CRPPR-targeted particles, the radioisotope density in the heart averaged 44 +/- 9% injected dose/gram of tissue, more than 30-fold higher than in skeletal muscle. The rapid and efficient targeting of these particles can be exploited in drug and gene delivery systems and with dynamic positron emission tomography provides a model system to optimize targeting of engineered particles.