Preclinical evaluation of somatostatin analogues for breast cancer imaging using positron emission tomography.

Somatostatin receptors (sstrs) are G-protein coupled receptors that modulate hormone secretions. Their overexpression on neuroendocrine tumours (NETs) has enabled successful imaging of these cancers with radioactive peptides using positron emission tomography. Luminal A breast cancers also overexpress sstr, but with a lower and more heterogeneous density than NETs. Recently, several authors demonstrated higher tumour binding with sstr antagonists compared to agonists. Antagonists are hypothesized to bind to target receptors in multiple configurations, thus labeling more binding sites than high-affinity agonists. This property could result in better visualization of tumours with lower sstr density, such as breast cancers. We hypothesized that the somatostatin agonist peptides TOC and TATE and antagonist peptide JR11 could be radiolabeled with ⁶⁸Ga and ¹⁸F, and that antagonist radiotracers will show higher tumour uptake when evaluated in vivo using the human breast cancer xenograft ZR-75-1. We synthesized the literature compounds ⁶⁸Ga-DOTATOC, ⁶⁸Ga-DOTATATE and antagonist ⁶⁸Ga-NODAGA-JR11 and evaluated binding affinity and tumour uptake. The agonist ⁶⁸Ga-DOTATOC had the highest tumour uptake (18.4 ± 2.87 %ID/g), and the antagonist ⁶⁸Ga-NODAGA-JR11, the lowest (12.1 ± 0.78 %ID/g). We radiolabeled the same peptide analogues with ¹⁸F using the easy and robust AmBF₃ method. ¹⁸F is an excellent isotope for peptide radiolabeling, having low positron energy (634 keV), a half-life well-matched to peptide kinetics (110 min), and an established cyclotron production method. We synthesized the compounds ¹⁸F-AmBF₃-TOC, ¹⁸F-AmBF3-TATE and ¹⁸F-AmBF3-JR11 and found that all were kinetically stable (determined by in vivo plasma stability studies) and successfully visualized the tumour with high intensity and contrast at both 1 hour and 2 hours post-injection (p.i.). At 1 h p.i. uptake means were between 10.0 – 13.4 %ID/g for all three radiotracers, and differences were not statistically significant. Imaging with ¹⁸F- and ⁶⁸Ga-labeled JR11 antagonists did not confer any additional advantages compared to agonists in this tumour model. However, the high tumour uptake of both ⁶⁸Ga- and ¹⁸F-labeled antagonists is striking, despite their significantly poorer binding affinity (inhibition contrast (Ki) > 18 nM) compared to agonist (Ki < 1.5 nM). With modifications to improve affinity, antagonist somatostatin analogues may be very promising for breast cancer visualization.Medicine, Faculty ofGraduat

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.Medicine, Faculty ofNon UBCRadiology, Department ofReviewedFacult

Additional file 1: Figure S1. of Unique depot formed by an oil based vaccine facilitates active antigen uptake and provides effective tumour control

Immune responses of DPX-formulated vaccine up to 50 days. HLA-A2 transgenic mice (HHD-DR1) received a single subcutaneous immunization with 50 uL of DPX-Survivac in the right flank. Groups of mice (n = 5) were terminated 8, 22 and 50 days after immunization and IFN-γ ELISPOT performed using lymph node cells isolated from the right inguinal lymph node. Cells were stimulated with syngeneic dendritic cells loaded with no peptide (empty), irrelevant peptide (ALMEQQHYV), or SurA2.M (LMLGEFLKL). (DOCX 40 kb

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 (<i>K</i>_i = 23.4–32.3 nM), while [Lys­(Ga-DOTA)⁴, Har⁶]­BVD15 (P05067) showed poor binding affinity (<i>K</i>_i > 1000 nM). In addition, CCZ01055 exhibited low binding affinity (<i>K</i>_i > 1000 nM) to Y2R and Y4R, demonstrating its selectivity to Y1R. The former three peptides showed improved <i>in vitro</i> 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

<i>p</i>‑NO₂–Bn–H₄neunpa and H₄neunpa–Trastuzumab: Bifunctional Chelator for Radiometalpharmaceuticals and ¹¹¹In Immuno-Single Photon Emission Computed Tomography Imaging

Potentially nonadentate (N₅O₄) bifunctional chelator <i>p</i>-SCN–Bn–H₄neunpa and its immunoconjugate H₄neunpa–trastuzumab for ¹¹¹In radiolabeling are synthesized. The ability of <i>p</i>-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 (<i>p</i>M) values. In vitro human serum stability assays have demonstrated both ¹¹¹In complexes to have high stability over 5 days. Mouse biodistribution of [¹¹¹In]­[In­(<i>p</i>-NO₂–Bn–neunpa)]⁻, compared to that of [¹¹¹In]­[In­(<i>p</i>-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 <i>p</i>-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