Pilot-phase PET/CT study targeting integrin αvß6 in pancreatic cancer patients using the cystine-knot peptide-based 18F-FP-R01-MG-F2.

PURPOSE: A novel cystine-knot peptide-based PET radiopharmaceutical, 18F-FP-R01-MG-F2 (knottin), was developed to selectively bind to human integrin αvß6 which is overexpressed in pancreatic cancer. The purpose of this study is to evaluate the safety, biodistribution, dosimetry, and lesion uptake of 18F-FP-R01-MG-F2 in patients with pancreatic cancer. METHODS: Fifteen patients (6 men, 9 women) with histologically confirmed pancreatic cancer were prospectively enrolled and underwent knottin PET/CT between March 2017 and February 2021 (ClinicalTrials.gov Identifier NCT02683824). Vital signs and laboratory results were collected before and after the imaging scans. Maximum standardized uptake values (SUVmax) and mean SUV (SUVmean) were measured in 24 normal tissues and pancreatic cancer lesions for each patient. From the biodistribution data, the organ doses and whole-body effective dose were calculated using OLINDA/EXM software. RESULTS: There were no significant changes in vital signs or laboratory values that qualified as adverse events or serious adverse events. At 1 h post-injection, areas of high 18F-FP-R01-MG-F2 uptake included the pituitary gland, stomach, duodenum, kidneys, and bladder (average SUVmean: 9.7-14.5). Intermediate uptake was found in the normal pancreas (average SUVmean: 4.5). Mild uptake was found in the lungs and liver (average SUVmean < 1.0). The effective dose was calculated to be 2.538 × 10-2 mSv/MBq. Knottin PET/CT detected all known pancreatic tumors in the 15 patients, although it did not detect small peri-pancreatic lymph nodes of less than 1 cm in short diameter in two of three patients who had lymph node metastases at surgery. Knottin PET/CT detected distant metastases in the lungs (n = 5), liver (n = 4), and peritoneum (n = 2), confirmed by biopsy and/or contrast-enhanced CT. CONCLUSION: 18F-FP-R01-MG-F2 is a safe PET radiopharmaceutical with an effective dose comparable to other diagnostic agents. Evaluation of the primary pancreatic cancer and distant metastases with 18F-FP-R01-MG-F2 PET is feasible, but larger studies are required to define the role of this approach. TRIAL REGISTRATION: NCT02683824.

SPECT/CT Imaging, Biodistribution and Radiation Dosimetry of a 177Lu-DOTA-Integrin αvß6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin αvß6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin αvß6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin αvß6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC. METHODS: The expression of integrin αvß6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model. RESULTS: RT-qPCR and immunofluorescence results showed high expression of integrin αvß6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin αvß6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 ± 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 ± 2.5 %IA/g, declining much more rapidly than in tumors. CONCLUSION: 177Lu-DOTA-integrin αvß6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

Evaluation of integrin αvß6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvß6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvß6.

Imaging Tumor Oxidative Stress with Surface Enhanced Raman Scattering Gold Nanoparticles.

We synthesized a new surface enhanced Raman scattering nanoparticle (SERS NP) which can detect reactive oxygen species (ROS) and thus changes in oxidative stress (OS). Our SERS NP was synthesized using a gold nanoparticle (AuNP) core which was then coated with a dihydrorhodamine (DHR123) Raman layer. In the presence of ROS, DHR123 is converted to rhodamine123 (Rd123) which has a distinct Raman fingerprint. Next, AuNP-DHR123 were encapsulated in a mesoporous-SiO2 shell to help appose DHR123 to the AuNP core. Finally, the AuNP-DHR123-mesoporous-SiO2 was functionalized with cystine knot peptides that target integrin αvß6. Our SERS NP was initially optimized in vitro using solutions containing reactive oxygen species as well as human cancer cell lines. Finally, in a xenograft animal model, we demonstrated the in vivo ability of our SERS NP to target a tumor, as well as provide a reading of the amount of OS within the tumor.

Development and Preclinical Validation of a Cysteine Knottin Peptide Targeting Integrin αvß6 for Near-infrared Fluorescent-guided Surgery in Pancreatic Cancer.

Purpose: Intraoperative near-infrared fluorescence (NIRF) imaging could help stratification for the proper primary treatment for patients with pancreatic ductal adenocarcinoma (PDAC), and achieve complete resection, as it allows visualization of cancer in real time. Integrin αvß6, a target specific for PDAC, is present in >90% of patients, and is able to differentiate between pancreatitis and PDAC. A clinically translatable αvß6-targeting NIRF agent was developed, based on a previously developed cysteine knottin peptide for PET imaging, R01-MG, and validated in preclinical mouse models.Experimental Design: The applicability of the agent was tested for cell and tissue binding characteristics using cell-based plate assays, subcutaneous, and orthotopic pancreatic models, and a transgenic mouse model of PDAC development (Pdx1-Cretg/+;KRasLSL G12D/+;Ink4a/Arf-/-). IRDye800CW was conjugated to R01-MG in a 1:1 ratio. R01-MG-IRDye800, was compared with a control peptide and IRDye800 alone.Results: In subcutaneous tumor models, a significantly higher tumor-to-background ratio (TBR) was seen in BxPC-3 tumors (2.5 ± 0.1) compared with MiaPaCa-2 (1.2 ± 0.1; P < 0.001), and to the control peptide (1.6 ± 0.4; P < 0.005). In an orthotopic tumor model, tumor-specific uptake of R01-MG-IRDye800 was shown compared with IRDye800 alone (TBR 2.7 vs. 0.86). The fluorescent signal in tumors of transgenic mice was significantly higher, TBR of 3.6 ± 0.94, compared with the normal pancreas of wild-type controls, TBR of 1.0 ± 0.17 (P < 0.001).Conclusions: R01-MG-IRDye800 shows specific targeting to αvß6, and holds promise as a diagnostic and therapeutic tool to recognize PDAC for fluorescence-guided surgery. This agent can help improve the stratification of patients for a potentially curative, margin-negative resection. Clin Cancer Res; 24(7); 1667-76. ©2018 AACR.

Thy1-Targeted Microbubbles for Ultrasound Molecular Imaging of Pancreatic Ductal Adenocarcinoma.

Purpose: To engineer a dual human and murine Thy1-binding single-chain-antibody ligand (Thy1-scFv) for contrast microbubble-enhanced ultrasound molecular imaging of pancreatic ductal adenocarcinoma (PDAC).Experimental Design: Thy1-scFv were engineered using yeast-surface-display techniques. Binding to soluble human and murine Thy1 and to Thy1-expressing cells was assessed by flow cytometry. Thy1-scFv was then attached to gas-filled microbubbles to create MBThy1-scFv Thy1 binding of MBThy1-scFv to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MBscFv-scrambled and MBNon-targeted were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice in vivoResults: Thy1-scFv had a KD of 3.4 ± 0.36 nmol/L for human and 9.2 ± 1.7 nmol/L for murine Thy1 and showed binding to both soluble and cellularly expressed Thy1. MBThy1-scFv was attached to Thy1 with high affinity compared with negative control microbubbles (P < 0.01) as assessed by flow cytometry. Similarly, flow-chamber studies showed significantly (P < 0.01) higher binding of MBThy1-scFv (3.0 ± 0.81 MB/cell) to Thy1-expressing cells than MBscFv-scrambled (0.57 ± 0.53) and MBNon-targeted (0.43 ± 0.53). In vivo ultrasound molecular imaging using MBThy1-scFv demonstrated significantly higher signal (P < 0.01) in both orthotopic (5.32 ± 1.59 a.u.) and transgenic PDAC (5.68 ± 2.5 a.u.) mice compared with chronic pancreatitis (0.84 ± 0.6 a.u.) and normal pancreas (0.67 ± 0.71 a.u.). Ex vivo immunofluorescence confirmed significantly (P < 0.01) increased Thy1 expression in PDAC compared with chronic pancreatitis and normal pancreas tissue.Conclusions: A dual human and murine Thy1-binding scFv was designed to generate contrast microbubbles to allow PDAC detection with ultrasound. Clin Cancer Res; 24(7); 1574-85. ©2018 AACR.

A Cystine Knot Peptide Targeting Integrin αvß6 for Photoacoustic and Fluorescence Imaging of Tumors in Living Subjects.

Photoacoustic imaging is a nonionizing biomedical imaging modality with higher resolution and imaging depth than fluorescence imaging, which has greater sensitivity. The combination of the 2 imaging modalities could improve the detection of cancer. Integrin αvß6 is a cell surface marker overexpressed in many different cancers. Here, we report the development and evaluation of a dye-labeled cystine knot peptide, which selectively recognizes integrin αvß6 with high affinity, for photoacoustic and fluorescence imaging. The new dual-modality probe may find clinical application in cancer diagnosis and intraoperative imaging of integrin αvß6-positive tumors. METHODS: An engineered cystine knot peptide, R01, that recognizes integrin αvß6 was labeled with Atto 740 (A740) and evaluated for its specific cell uptake and its sensitivity threshold. A740-R01 was injected via the tail vein into nude mice xenografted with A431 (integrin αvß6-positive) or 293T (integrin αvß6-negative) tumors. Photoacoustic and fluorescence scans of tumors were acquired before and at 0.5, 1, 2, and 4 h after injection of A740-R01. Dynamic photoacoustic scans of various normal organs were also acquired. Ex vivo fluorescence imaging of tissues was performed 1 h after injection. RESULTS: The A740-R01 demonstrated integrin αvß6-dependent binding to A431 cells in culture. Sensitivity studies indicated that the probe may potentially detect lesions as small as 1 or 6 mm3 by fluorescence or photoacoustic imaging, respectively. The photoacoustic and fluorescence signals of A431 xenografts at 1 h after injection were 1.87 ± 0.25 arbitrary units (AU) and 8.27 ± 0.87 AU, respectively. Target specificity was confirmed by low tumor uptake in 293T tumors at 1 h after injection (1.07 ± 0.15 AU and 1.10 ± 0.14 AU for photoacoustic and fluorescence signals, respectively). A740-R01 exhibited hepatobiliary clearance marked by high uptake in the liver, spleen, and intestine but low uptake in the kidneys. CONCLUSION: A740-R01 specifically targeted integrin αvß6 with low nanomolar affinity. A740-R01 was able to detect integrin αvß6 both in vitro and in vivo by photoacoustic and fluorescence imaging. A740-R01 is able to detect αvß6-positive tumors in living subjects and may have clinical application in cancer diagnosis and real-time image-guided surgery.

Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2.

Monobodies are binding scaffold proteins originating from a human fibronectin domain III (Fn3) scaffold that can be easily engineered with specificity and affinity. Human EphA2 (hEphA2) is an early detection marker protein for various tumors including lung, breast, and colon cancer. In this study, we isolated two hEphA2-specific monobodies (E1 and E10) by screening a yeast surface display library. They showed the same amino acid sequence except in the DE loop and had high affinity (~2 nM Kd) against hEphA2. E1 bound only hEphA2 and mEphA2, although it bound hEphA2 with an affinity 2-fold higher than that of mEphA2. However, E10 also bound the mEphA6 and mEphA8 homologs as well as hEphA2 and mEphA2. Thus, E1 but not E10 was highly specific for hEphA2. E1 specifically bound human cells and xenograft tumor tissues expressing hEphA on the cell surface. In vivo optical imaging showed strong targeting of Cy5.5-labeled E1 to mouse tumor tissue induced by PC3 cells, a human prostate cancer cell line that expresses a high level of hEphA2. In conclusion, the highly specific monobody E1 is useful as a hEphA2 probe candidate for in vivo diagnosis and therapy.

64Cu-Labeled Divalent Cystine Knot Peptide for Imaging Carotid Atherosclerotic Plaques.

UNLABELLED: The rupture of vulnerable atherosclerotic plaques that lead to stroke and myocardial infarction may be induced by macrophage infiltration and augmented by the expression of integrin αvß3. Indeed, atherosclerotic angiogenesis may be a promising marker of inflammation. In this study, an engineered integrin αvß3-targeting PET probe, (64)Cu-NOTA-3-4A, derived from a divalent knottin miniprotein was evaluated in a mouse model for carotid atherosclerotic plaques. METHODS: Atherosclerotic plaques in BALB/C mice, maintained on a high-fat diet, were induced with streptozotocin injection and carotid artery ligation and verified by MR imaging. Knottin 3-4A was synthesized by solid-phase peptide synthesis chemistry and coupled to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) before radiolabeling with (64)Cu. PET probe stability in mouse serum was evaluated. Mice with carotid atherosclerotic plaques were injected via the tail vein with (64)Cu-NOTA-3-4A or (18)F-FDG, followed by small-animal PET/CT imaging at different time points. Receptor targeting specificity of the probe was verified by coinjection of c(RGDyK) administered in molar excess. Subsequently, carotid artery dissection and immunofluorescence staining were performed to evaluate target expression. RESULTS: (64)Cu-NOTA-3-4A was synthesized in high radiochemical purity and yield and demonstrated molecular stability in both phosphate-buffered saline and mouse serum at 4 h. Small-animal PET/CT showed that (64)Cu-NOTA-3-4A accumulated at significantly higher levels in the neovasculature of carotid atherosclerotic plaques (7.41 ± 1.44 vs. 0.67 ± 0.23 percentage injected dose/gram, P < 0.05) than healthy or normal vessels at 1 h after injection. (18)F-FDG also accumulated in atherosclerotic lesions at 0.5 and 1 h after injection but at lower plaque-to-normal tissue ratios than (64)Cu-NOTA-3-4A. For example, plaque-to-normal carotid artery ratios for (18)F-FDG and (64)Cu-NOTA-3-4A at 1 h after injection were 3.75 and 14.71 (P < 0.05), respectively. Furthermore, uptake of (64)Cu-NOTA-3-4A in atherosclerotic plaques was effectively blocked (∼90% at 1 h after injection) by coinjection of c(RGDyK). Immunostaining confirmed integrin αvß3 expression in both the infiltrating macrophages and the neovasculature of atherosclerotic plaques. CONCLUSION: (64)Cu-NOTA-3-4A demonstrates specific accumulation in carotid atherosclerotic plaques in which macrophage infiltration and angiogenesis are responsible for elevated integrin αvß3 levels. Therefore, (64)Cu-NOTA-3-4A may demonstrate clinical utility as a PET probe for atherosclerosis imaging or for the evaluation of therapies used to treat atherosclerosis.

Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope.

PURPOSE: To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. MATERIALS AND METHODS: Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. RESULTS: The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. CONCLUSION: Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.

A radiofluorinated divalent cystine knot peptide for tumor PET imaging.

A divalent knottin containing two separate integrin binding epitopes (RGD) in the adjacent loops, 3-4A, was recently developed and reported in our previous publication. In the current study, 3-4A was radiofluorinated with a 4-nitrophenyl 2-(18)F-fluoropropinate ((18)F-NFP) group and the resulting divalent positron emission tomography (PET) probe, (18)F-FP-3-4A, was evaluated as a novel imaging probe to detect integrin αvß3 positive tumors in living animals. Knottin 3-4A was synthesized by solid phase peptide synthesis, folded, and site-specifically conjugated with (18/19)F-NFP to produce the fluorinated peptide (18/19)F-fluoropropinate-3-4A ((18/19)F-FP-3-4A). The stability of (18)F-FP-3-4A was tested in both phosphate buffered saline (PBS) buffer and mouse serum. Cell uptake assays of the radiolabeled peptides were performed using U87MG cells. In addition, small animal PET imaging and biodistribution studies of (18)F-FP-3-4A were performed in U87MG tumor-bearing mice. The receptor targeting specificity of the radiolabeled peptide was also verified by coinjecting the probe with a blocking peptide cyclo(RGDyK). Our study showed that (18)F-FP-3-4A exhibited excellent stability in PBS buffer (pH 7.4) and mouse serum. Small animal PET imaging and biodistribution data revealed that (18)F-FP-3-4A exhibited rapid and good tumor uptake (3.76 ± 0.59% ID/g and 2.22 ± 0.62% ID/g at 0.5 and 1 h, respectively). (18)F-FP-3-4A was rapidly cleared from the normal tissues, resulting in excellent tumor-to-normal tissue contrasts. For example, liver uptake was only 0.39 ± 0.07% ID/g and the tumor to liver ratio was 5.69 at 1 h p.i. Furthermore, coinjection of cyclo(RGDyK) with (18)F-FP-3-4A significantly inhibited tumor uptake (0.41 ± 0.12 vs 1.02 ± 0.19% ID/g at 2.5 h) in U87MG xenograft models, demonstrating specific accumulation of the probe in the tumor. In summary, the divalent probe (18)F-FP-3-4A is characterized by rapid and high tumor uptake and excellent tumor-to-normal tissue ratios. (18)F-FP-3-4A is a highly promising knottin based PET probe for translating into clinical imaging of tumor angiogenesis.

99mTc-labeled cystine knot peptide targeting integrin αvß6 for tumor SPECT imaging.

Integrin αvß6 is overexpressed in a variety of cancers, and its expression is often associated with poor prognosis. Therefore, there is a need to develop affinity reagents for noninvasive imaging of integrin αvß6 expression since it may provide early cancer diagnosis, more accurate prognosis, and better treatment planning. We recently engineered and validated highly stable cystine knot peptides that selectively bind integrin αvß6 with no cross-reactivity to integrins αvß5, α5ß1, or αvß3, also known to be overexpressed in many cancers. Here, we developed a single photon emission computed tomography (SPECT) probe for imaging integrin αvß6 positive tumors. Cystine knot peptide, S02, was first conjugated with a single amino acid chelate (SAAC) and labeled with [(99m)Tc(H2O)3(CO)3](+). The resulting probe, (99m)Tc-SAAC-S02, was then evaluated by in vitro cell uptake studies using two αvß6 positive cell lines (human lung adenocarcinoma cell line HCC4006 and pancreatic cancer cell line BxPC-3) and two αvß6 negative cell lines (human lung adenocarcinoma cell line H838 and human embryonic kidney cell line 293T). Next, SPECT/CT and biodistribution studies were performed in nude mice bearing HCC4006 and H838 tumor xenografts to evaluate the in vivo performance of (99m)Tc-SAAC-S02. Significant differences in the uptake of (99m)Tc-SAAC-S02 were observed in αvß6 positive vs negative cells (P < 0.05). Biodistribution and small animal SPECT/CT studies revealed that (99m)Tc-SAAC-S02 accumulated to moderate levels in antigen positive tumors (∼2% ID/g at 1 and 6 h postinjection, n = 3 or 4/group). Moreover, the probe demonstrated tumor-to-background tissue ratios of 6.81 ± 2.32 (tumor-to-muscle) and 1.63 ± 0.18 (tumor-to-blood) at 6 h postinjection in αvß6 positive tumor xenografts. Co-incubation of the probe with excess amount of unlabeled S02 as a blocking agent demonstrated significantly reduced tumor uptake, which is consistent with specific binding to the target. Renal filtration was the main route of clearance. In conclusion, knottin peptides are excellent scaffolds for which to develop highly stable imaging probes for a variety of oncological targets. (99m)Tc-SAAC-S02 demonstrates promise for use as a SPECT agent to image integrin αvß6 expression in living systems.

18F-fluorobenzoate-labeled cystine knot peptides for PET imaging of integrin αvß6.

UNLABELLED: Integrin αvß6 is a cell surface receptor minimally expressed by healthy tissue but elevated in lung, colon, skin, ovarian, cervical, and pancreatic cancers. A molecular PET agent for integrin αvß6 could provide significant clinical utility by facilitating both cancer staging and treatment monitoring to more rapidly identify an effective therapeutic approach. METHODS: Here, we evaluated 2 cystine knot peptides, R01 and S02, previously engineered with a 3-6 nM affinity for integrin αvß6, for (18)F radiolabeling and PET imaging of BxPC3 pancreatic adenocarcinoma xenografts in mice. Cystine knot peptides were labeled with N-succinimidyl-4-(18)F-fluorobenzoate and evaluated for binding affinity and serum stability. Peptides conjugated with (18)F-fluorobenzoate (2-3 MBq) were injected via the tail vein into nude mice xenografted with BxPC3 (integrin αvß6-positive) or 293 (integrin αvß6-negative) tumors. Small-animal PET scans were acquired at 0.5, 1, and 2 h after injection. Ex vivo γ-counting of dissected tissues was performed at 0.5 and 2 h. RESULTS: (18)F-fluorobenzoate peptides were produced in 93% ((18)F-fluorobenzoate-R01) and 99% ((18)F-fluorobenzoate-S02) purity. (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02 had affinities of 1.1 ± 0.2 and 0.7 ± 0.4 nM, respectively, and were 87% and 94%, respectively, stable in human serum at 37°C for 2 h. (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02 exhibited 2.3 ± 0.6 and 1.3 ± 0.4 percentage injected dose per gram (%ID/g), respectively, in BxPC3 xenografted tumors at 0.5 h (n = 4-5). Target specificity was confirmed by low tumor uptake in integrin αvß6-negative 293 tumors (1.4 ± 0.6 and 0.5 ± 0.2 %ID/g, respectively, for (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02; both P < 0.05; n = 3-4) and low muscle uptake (3.1 ± 1.0 and 2.7 ± 0.4 tumor to muscle for (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02, respectively). Small-animal PET data were corroborated by ex vivo γ-counting of dissected tissues, which demonstrated low uptake in nontarget tissues with only modest kidney uptake (9.2 ± 3.3 and 1.9 ± 1.2 %ID/g, respectively, at 2 h for (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02; n = 8). Uptake in healthy pancreas was low (0.3% ± 0.1% for (18)F-fluorobenzoate-R01 and 0.03% ± 0.01% for (18)F-fluorobenzoate-S02; n = 8). CONCLUSION: These cystine knot peptide tracers, in particular (18)F-fluorobenzoate-R01, show translational promise for molecular imaging of integrin αvß6 overexpression in pancreatic and other cancers.

Structure-activity relationships of 6-(aminomethylphenoxy)-benzoxaborole derivatives as anti-inflammatory agent.

A series of novel 6-(aminomethylphenoxy)benzoxaborole analogs was synthesized for the investigation of the structure-activity relationship of the inhibition of TNF-alpha, IL-1beta, and IL-6, from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compounds 9d and 9e showed potent activity against all three cytokines with IC50 values between 33 and 83nM. Chloro substituted analog 9e (AN3485) is considered to be a promising lead for novel anti-inflammatory agent with a favorable pharmacokinetic profile.

Boron-based phosphodiesterase inhibitors show novel binding of boron to PDE4 bimetal center.

We have used boron-based molecules to create novel, competitive, reversible inhibitors of phosphodiesterase 4 (PDE4). The co-crystal structure reveals a binding configuration which is unique compared to classical catechol PDE4 inhibitors, with boron binding to the activated water in the bimetal center. These phenoxybenzoxaboroles can be optimized to generate submicromolar potency enzyme inhibitors, which inhibit TNF-α, IL-2, IFN-γ, IL-5 and IL-10 activities in vitro and show safety and efficacy for topical treatment of human psoriasis. They provide a valuable new route for creating novel potent anti-PDE4 inhibitors.

111In-labeled cystine-knot peptides based on the Agouti-related protein for targeting tumor angiogenesis.

Agouti-related protein (AgRP) is a 4-kDa cystine-knot peptide of human origin with four disulfide bonds and four solvent-exposed loops. The cell adhesion receptor integrin α(v)ß(3) is an important tumor angiogenesis factor that determines the invasiveness and metastatic ability of many malignant tumors. AgRP mutants have been engineered to bind to integrin α(v)ß(3) with high affinity and specificity using directed evolution. Here, AgRP mutants 7C and 6E were radiolabeled with (111)In and evaluated for in vivo targeting of tumor integrin α(v)ß(3) receptors. AgRP peptides were conjugated to the metal chelator 1, 4, 7, 10-tetra-azacyclododecane- N, N', N″, N'''-tetraacetic acid (DOTA) and radiolabeled with (111)In. The stability of the radiopeptides (111)In-DOTA-AgRP-7C and (111)In-DOTA-AgRP-6E was tested in phosphate-buffered saline (PBS) and mouse serum, respectively. Cell uptake assays of the radiolabeled peptides were performed in U87MG cell lines. Biodistribution studies were performed to evaluate the in vivo performance of the two resulting probes using mice bearing integrin-expressing U87MG xenograft tumors. Both AgRP peptides were easily labeled with (111)In in high yield and radiochemical purity (>99%). The two probes exhibited high stability in phosphate-buffered saline and mouse serum. Compared with (111)In-DOTA-AgRP-6E, (111)In-DOTA-AgRP-7C showed increased U87MG tumor uptake and longer tumor retention (5.74 ± 1.60 and 1.29 ± 0.02%ID/g at 0.5 and 24 h, resp.), which was consistent with measurements of cell uptake. Moreover, the tumor uptake of (111)In-DOTA-AgRP-7C was specifically inhibited by coinjection with an excess of the integrin-binding peptidomimetic c(RGDyK). Thus, (111)In-DOTA-AgRP-7C is a promising probe for targeting integrin α(v)ß(3) positive tumors in living subjects.

Use of (64)Cu-labeled fibronectin domain with EGFR-overexpressing tumor xenograft: molecular imaging.

PURPOSE: To assess the ability of an engineered epidermal growth factor receptor (EGFR)-binding fibronectin domain to serve as a positron emission tomographic (PET) probe for molecular imaging of EGFR in a xenograft mouse model. MATERIALS AND METHODS: An EGFR-binding fibronectin domain (fibronectin abbreviated to Fn when bound) was site-specifically labeled with copper 64 ((64)Cu) (8 MBq/nmol). Copper 64-Fn binding was tested in cell cultures with varying EGFR expression. Stability in human and mouse serum was measured in vitro. Animal experiments were approved by the Stanford University Institutional Animal Care and Use Committee. Copper 64-Fn (approximately 2 MBq) was used for PET in mice (n = 5) bearing EGFR-overexpressing xenografted tumors (approximately 5-10 mm in diameter). Results of tomography were compared with those of ex vivo gamma counting of dissected tissues. Statistical analysis was performed with t tests and adjustment for multiple comparisons. RESULTS: Copper 64-Fn exhibited EGFR-dependent binding to multiple cell lines in culture. The tracer was stable for 24 hours in human and mouse serum at 37°C. The tracer exhibited good tumor localization (3.4% injected dose [ID]/g ± 1.0 [standard deviation] at 1 hour), retention (2.7% ID/g ± 0.6 at 24 hours), and specificity (8.6 ± 3.0 tumor-to-muscle ratio, 8.9 ± 4.7 tumor-to-blood ratio at 1 hour). Specific targeting was verified with low localization to low-expressing MDA-MB-435 tumors (0.7% ID/g ± 0.8 at 1 hour, P = .018); specificity was further demonstrated, as a nonbinding control fibronectin had low localization to EGFR-overexpressing xenografts (0.8% ID/g ± 0.2 at 1 hour, P = .013). CONCLUSION: The stability, low background, and target-specific tumor uptake and retention of the engineered fibronectin domain make it a promising EGFR molecular imaging agent. More broadly, it validates the fibronectin domain as a potential scaffold for a generation of various molecular imaging agents.

Pharmacokinetically stabilized cystine knot peptides that bind alpha-v-beta-6 integrin with single-digit nanomolar affinities for detection of pancreatic cancer.

PURPOSE: Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin α(v)ß(6), a cell surface receptor being evaluated as a novel clinical biomarker. EXPERIMENTAL DESIGN: To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3-6 nmol/L) to integrin α(v)ß(6). The binders do not cross-react with related integrin α(v)ß(5), integrin α(5)ß(1), or tumor-angiogenesis-associated integrin, α(v)ß(3). RESULTS: Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin α(v)ß(6). Clinically relevant tumor-to-muscle ratios of 7.7 ± 2.4 to 11.3 ± 3.0 were achieved within 1 hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting α(v)ß(6)-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3). CONCLUSIONS: We have engineered highly stable cystine knot peptides with potent and specific integrin α(v)ß(6)-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging.