Toward tryptathionine-stapled one-bead-one-compound (OBOC) libraries: solid phase synthesis of a bioactive octretoate analog.

New somatostatin analogs are highly desirable for diagnosing and treating neuroendocrine tumors (NETs). Here we describe the solid-phase synthesis of a new octreotate (TATE) analog where the disulfide bond is replaced with a tryptathionine (Ttn) staple as part of an effort to prototyping a one-bead-one-compound (OBOC) library of Ttn-stapled peptides. Library design provides the potential for on- and off-bead screening. To validate our method, we labelled Ttn-TATE with a fluorescent dye to demonstrate binding to soluble somatostatin receptor subtype-2 and staining of Ar42J rat prostate cancer cells. By exploring this staple in the context of a ligand of known affinity, this method paves the way for an OBOC library construction of bioactive octreotate analogs and, more broadly speaking, tryptathionine-staped peptide macrocycles.

Unique depot formed by an oil based vaccine facilitates active antigen uptake and provides effective tumour control.

BACKGROUND: Oil emulsions are commonly used as vaccine delivery platforms to facilitate slow release of antigen by forming a depot at the injection site. Antigen is trapped in the aqueous phase and as the emulsion degrades in vivo the antigen is passively released. DepoVax™ is a unique oil based delivery system that directly suspends the vaccine components in the oil diluent that forces immune cells to actively take up components from the formulation in the absence of passive release. The aim of this study was to use magnetic resonance imaging (MRI) with additional biological markers to evaluate and understand differences in clearance between several different delivery systems used in peptide-based cancer vaccines. METHODS: C57BL/6 mice were implanted with a cervical cancer model and vaccinated 5 days post-implant with either DepoVax (DPX), a water-in-oil emulsion (w/o), a squalene oil-in-water emulsion (squal o/w) or a saponin/liposome emulsion (sap/lip) containing iron oxide-labeled targeted antigen. MRI was then used to monitor antigen clearance, the site of injection, tumour and inguinal lymph node volumes and other gross anatomical changes. HLA-A2 transgenic mice were also vaccinated to evaluate immune responses of human directed peptides. RESULTS: We demonstrated differences in antigen clearance between DPX and w/o both in regard to how quickly the antigen was cleared and the pattern in which it was cleared. We also found differences in lymph node responses between DPX and both squal o/w and sap/lip. CONCLUSIONS: These studies underline the unique mechanism of action of this clinical stage vaccine delivery system.

Estrogen Receptor ß Is a Novel Target in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a devastating disease characterized by poor patient outcome and suboptimal chemotherapeutics. Here, a high-throughput screen identified diosmetin, a citrus flavonoid, with anti-AML activity. Diosmetin imparted selective toxicity against leukemia and leukemia stem cells in vitro and in vivo with no effect on normal hematopoietic stem cells. Mechanistically, we demonstrated that diosmetin targets estrogen receptor (ER) ß. ERß expression conferred cell sensitivity, as patient-derived AML cells with high levels of ERß were sensitive, whereas cells with low ERß were insensitive to diosmetin. Knockdown of ERß confirmed resistance, whereas overexpression enhanced sensitivity to diosmetin, which was demonstrated to be mediated by reactive oxygen species signaling. In summary, these studies highlight targeting of ERß with diosmetin as a potential novel therapeutic strategy for the treatment of AML. Mol Cancer Ther; 16(11); 2618-26. ©2017 AACR.

p-NO2-Bn-H4neunpa and H4neunpa-Trastuzumab: Bifunctional Chelator for Radiometalpharmaceuticals and 111In Immuno-Single Photon Emission Computed Tomography Imaging.

Potentially nonadentate (N5O4) bifunctional chelator p-SCN-Bn-H4neunpa and its immunoconjugate H4neunpa-trastuzumab for 111In radiolabeling are synthesized. The ability of p-SCN-Bn-H4neunpa and H4neunpa-trastuzumab to quantitatively radiolabel 111InCl3 at an ambient temperature within 15 or 30 min, respectively, is presented. Thermodynamic stability determination with In3+, Bi3+, and La3+ resulted in high conditional stability constant (pM) values. In vitro human serum stability assays have demonstrated both 111In complexes to have high stability over 5 days. Mouse biodistribution of [111In][In(p-NO2-Bn-neunpa)]-, compared to that of [111In][In(p-NH2-Bn-CHX-A″-diethylenetriamine pentaacetic acid (DTPA))]2-, at 1, 4, and 24 h shows fast clearance of both complexes from the mice within 24 h. In a second mouse biodistribution study, the immunoconjugates 111In-neunpa-trastuzumab and 111In-CHX-A″-DTPA-trastuzumab demonstrate a similar distribution profile but with slightly lower tumor uptake of 111In-neunpa-trastuzumab compared to that of 111In-CHX-A″-DTPA-trastuzumab. These results were also confirmed by immuno-single photon emission computed tomography (immuno-SPECT) imaging in vivo. These initial investigations reveal the acyclic bifunctional chelator p-SCN-Bn-H4neunpa to be a promising chelator for 111In (and other radiometals) with high in vitro stability and also show H4neunpa-trastuzumab to be an excellent 111In chelator with promising biodistribution in mice.

Preclinical Melanoma Imaging with 68Ga-Labeled α-Melanocyte-Stimulating Hormone Derivatives Using PET.

It is estimated that melanoma accounted for 76,380 new cases and 10,130 deaths in the United States in 2016. The melanocortin 1 receptor (MC1R) is highly expressed in the vast majority of melanomas, which makes it an attractive target for molecular imaging and radionuclide therapy. Lactam bridge-cyclized α-melanocyte-stimulating hormone (Ac-Nle4-cyclo[Asp5-His-D-Phe7-Arg-Trp-Lys10]-NH2, or Nle-CycMSHhex) analogues have been successfully developed and studied for MC1R-targeted imaging, predominantly with single-photon emission computed tomography (SPECT). The goal of this study was to design and evaluate novel peptides for melanoma imaging with positron emission tomography (PET). We designed and synthesized three peptides, DOTA-PEG2-Nle-CycMSHhex (CCZ01047), DOTA-4-amino-(1-carboxymethyl) piperidine (Pip)-Nle-CycMSHhex (CCZ01048), and DOTA-Pip-Pip-Nle-CycMSHhex (CCZ01056). All three peptides exhibited high binding affinity to MC1R with sub-nanomolar Ki values, rapid internalization into B16F10 melanoma cells and high in vivo stability with more than 93% remaining intact at 15 min post-injection (p.i.) in blood plasma. All three 68Ga-labeled tracers produced high contrast PET images in C57BL/6J mice bearing B16F10 tumors, and their respective tumor uptakes were 8.0 ± 3.0, 12.3 ± 3.3, and 6.5 ± 1.4 %ID/g at 1 h p.i. Minimal normal organ activity was observed at 1 h p.i., except for kidneys (5.1 ± 1.4, 4.7 ± 0.5, and 6.2 ± 2.0 %ID/g, respectively), and thyroid (4.1 ± 0.6 %ID/g for CCZ01047 and 2.4 ± 0.6 %ID/g for CCZ01048). Due to high accumulation at tumor sites and rapid background clearance of 68Ga-CCZ01048, we further evaluated it at 2 h p.i., and a tumor uptake of 21.9 ± 4.6 %ID/g was observed, with background activity further decreased. Exceptional image contrast was also achieved, i.e. tumor-to-blood, tumor-to-muscle, tumor-to-bone and tumor-to-kidney ratios were 96.4 ± 13.9, 210.9 ± 20.9, 39.6 ± 11.9 and 4.0 ± 0.9, respectively. A blocking study was also performed by co-injection of excess amount of non-radioactive Ga-coupled of CCZ01048, which confirmed that the tumor uptake was MC1R mediated. In conclusion, the introduction of a cationic Pip linker to Nle-CycMSHhex, CCZ01048, not only improved tumor uptake, but also generated high tumor-to-normal tissue contrast with PET imaging in a preclinical melanoma model. Therefore, CCZ01048 is a promising candidate for PET imaging of melanoma, and potentially as a theranostic agent for radionuclide therapy of melanoma when labeled with α or ß emitters.

2-18F-Fluoroethanol Is a PET Reporter of Solid Tumor Perfusion.

Solid tumor perfusion is a proven variable of interest for predicting cancer aggression and response to therapy. Current methods for noninvasively imaging tumor perfusion with PET are limited by restricted accessibility and short half-lives of perfusion radiotracers. This study presents 2-18F-fluoroethanol (2-18F-FEtOH) as a perfusion reporter that can distinguish between tumors of varying perfusion levels and can be applied to screening drugs that modify tumor perfusion. Methods: Uptake of 2-18F-FEtOH in 4T1 and 67NR murine mammary carcinoma tumors grown in mice was measured using ex vivo radiography as well as static and dynamic PET imaging. 2-18F-FEtOH uptake was directly compared with the 14C-iodoantipyrine perfusion reporter, and the perfusion-modifying drugs nicotinamide, pentoxifylline, and hydralazine were used to manipulate tumor perfusion before 2-18F-FEtOH quantification. Results: Uptake of 2-18F-FEtOH in 4T1 and 67NR tumors was consistent with known perfusion differences within and between these tumors. 2-18F-FEtOH uptake corresponded well with 14C-iodoantipyrine and reflected the tumor perfusion-modifying effects of each drug. Conclusion: 2-18F-FEtOH is a novel 18F-based radiotracer for investigating tumor perfusion with PET imaging. Quantification of 2-18F-FEtOH uptake can be used to distinguish between tumors of varying perfusion and to screen the efficacy of blood flow-modifying drugs for use as adjuvants to existing cancer therapies.

Evaluation of agonist and antagonist radioligands for somatostatin receptor imaging of breast cancer using positron emission tomography.

BACKGROUND: The somatostatin receptor subtype 2 (sstr2) is expressed on a majority of luminal breast cancers, however SPECT and scintigraphy imaging with agonistic sstr2 probes has been sub-optimal. High affinity antagonists can access more binding sites on the cell surface, resulting in higher tumor uptake and improved sensitivity. We compared the tumor uptake and biodistribution of the antagonist 68Ga-NODAGA-JR11 with two agonists 68Ga-DOTA-Tyr3-octreotide (68Ga-DOTATOC) and 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTATATE), in the human, sstr2-positive, luminal breast cancer model: ZR-75-1. RESULTS: Peptides were assayed for binding affinity using a filtration-based competitive assay to sstr2. natGa-DOTATOC and natGa-DOTATATE had excellent affinity (inhibition constant Ki: 0.9 ± 0.1 nM and 1.4 ± 0.3 nM respectively) compared to natGa-NODAGA-JR11 (25.9 ± 0.2 nM). The number of binding sites on ZR-75-1 cells was determined in vitro by saturation assays. Agonist 67/natGa-DOTATOC bound to 6.64 ± 0.39 × 104 sites/cells, which was 1.5-fold higher than 67/natGa-NODAGA-JR11 and 2.3-fold higher than 67/natGa-DOTATATE. All three 68Ga-labeled peptides were obtained in good decay-corrected radiochemical yield (61-68%) and were purified by high performance liquid chromatography to ensure high specific activity (137 - 281 MBq/nmol at the end of synthesis). NOD scid gamma mice bearing ZR-75-1 tumors were injected intravenously with the labeled peptides and used for PET/CT imaging and biodistribution at 1 h post-injection. We found that 68Ga-DOTATOC had the highest tumor uptake (18.4 ± 2.9%ID/g), followed by 68Ga-DOTATATE (15.2 ± 2.2%ID/g) and 68Ga-NODAGA-JR11 (12.2 ± 0.8%ID/g). Tumor-to-blood and tumor-to-muscle ratios were also higher for the agonists (>40 and >150 respectively), compared to the antagonist (15.6 ± 2.2 and 45.2 ± 11.6 respectively). CONCLUSIONS: The antagonist 68Ga-NODAGA-JR11 had the lowest tumor uptake and contrast compared to agonists 68Ga-DOTATOC and 68Ga-DOTATATE in ZR-75-1 xenografts.The main contributing factor to this result could be the use of an endogenously expressing cell line, which may differ from previously published transfected models in the number of low-affinity, antagonist-specific binding sites. The relative merit of agonists versus antagonists for sstr2 breast cancer imaging warrants further investigation, first in preclinical models with other sstr2-positive breast cancer xenografts, and ultimately in luminal breast cancer patients.

Targeting the Neuropeptide Y1 Receptor for Cancer Imaging by Positron Emission Tomography Using Novel Truncated Peptides.

The neuropeptide Y1 receptor (Y1R) is overexpressed in many human cancers, particularly breast cancer. Due to stability issues, limited success has been achieved for Y1R imaging agents, including full length and truncated neuropeptide Y (NPY) analogues. The goal of this study was to evaluate the possibility of using radiolabeled truncated NPY analogues to visualize Y1R expression in a preclinical model of Y1R-positive tumor. Four truncated NPY analogues were synthesized based on the sequence of [Pro30, Tyr32, Leu34]NPY(28-36), also known as BVD15. We substituted Tyr5 and Arg6 with unnatural amino acids aiming to enhance plasma stability while maintaining good receptor binding affinity to Y1R. In addition, we substituted Leu4 to Lys4 in order to conjugate via an optional linker the DOTA chelator for 68Ga labeling. Receptor binding affinity and plasma stability of these compounds were evaluated. Positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies were performed using immune-compromised mice bearing HEK293T::WT and HEK293T::hY1R tumors. [Lys(Ga-DOTA)4, Bip5]BVD15 (CCZ01035), [Lys(Ahx-Ga-DOTA)4, Bip5]BVD15 (CCZ01053), and [Lys(Pip-Ga-DOTA)4, Bip5]BVD15 (CCZ01055) demonstrated good binding affinity to Y1R (Ki = 23.4-32.3 nM), while [Lys(Ga-DOTA)4, Har6]BVD15 (P05067) showed poor binding affinity (Ki > 1000 nM). In addition, CCZ01055 exhibited low binding affinity (Ki > 1000 nM) to Y2R and Y4R, demonstrating its selectivity to Y1R. The former three peptides showed improved in vitro plasma stability of 7-16% remaining intact after 1 h incubation. PET/CT imaging and biodistribution studies for 68Ga-labeled CCZ01053, CCZ01035, and CCZ01055 showed that radioactivity was mainly cleared by the renal pathway, and HEK293T::hY1R tumors were clearly visualized with minimal background activity with the latter two. Of these two tracers, [68Ga]CCZ01055 provided lower kidney accumulation and higher contrast, i.e., average uptake ratios of Y1R tumor to wild type tumor, blood, and muscle are 3.87 ± 0.83, 4.12 ± 1.14, and 17.6 ± 4.64, respectively. Furthermore, Y1R tumor uptake with [68Ga]CCZ01055 was significantly reduced with coinjection of 100 µg of peptide YY, confirming the specificity of tumor accumulation was receptor mediated. We successfully developed the first Y1R-targeting truncated NPY analogues for PET imaging in a preclinical model, and [68Ga]CCZ01055 is a critical template for designing improved imaging agents to detect Y1R expressing cancers.

Using lymph node swelling as a potential biomarker for successful vaccination.

There is currently a lack of biomarkers to help properly assess novel immunotherapies at both the preclinical and clinical stages of development. Recent work done by our group indicated significant volume changes in the vaccine draining right lymph node (RLN) volumes of mice that had been vaccinated with DepoVaxTM, a lipid-based vaccine platform that was developed to enhance the potency of peptide-based vaccines. These changes in lymph node (LN) volume were unique to vaccinated mice.To better assess the potential of volumetric LN markers for multiple vaccination platforms, we evaluated 100 tumor bearing mice and assessed their response to vaccination with either a DepoVax based vaccine (DPX) or a water-in-oil emulsion (w/o), and compared them to untreated controls. MRI was used to longitudinally monitor LN and tumor volumes weekly over 4 weeks. We then evaluated changes in LN volumes occurring in response to therapy as a potential predictive biomarker for treatment success.We found that for both vaccine types, DPX and w/o, the %RLN volumetric increase over baseline and the ratio of RLN/LLN were strong predictors of successful tumor suppression (LLN is left inguinal LN). The area under the curve (AUC) was greatest, between 0.75-0.85, two (%RLN) or three (RLN/LLN) weeks post-vaccination. For optimized critical thresholds we found these biomarkers consistently had sensitivity >90% and specificity >70% indicating strong prognostic potential. Vaccination with DepoVax had a more pronounced effect on draining lymph nodes than w/o emulsion vaccines, which correlated with a higher anti-tumor activity in DPX-treated mice.