A radiofluorinated divalent cystine knot peptide for tumor PET imaging.
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ABSTRACT:
SUBMITTER: Jiang L
PROVIDER: S-EPMC4212002 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Integrin αvβ6 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 αvβ6 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 αvβ6 with no cross-reactivity to integrins αvβ5, α5β1, or αvβ3, also known to be overexpressed in many cancers. Here, we developed a single photon emission computed tomography (SPECT) probe for imaging integrin αvβ6 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 αvβ6 positive cell lines (human lung adenocarcinoma cell line HCC4006 and pancreatic cancer cell line BxPC-3) and two αvβ6 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 αvβ6 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 αvβ6 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 αvβ6 expression in living systems.
| S-EPMC3993876 | biostudies-other
Project description:Integrin ?v?6 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 ?v?6 could provide significant clinical utility by facilitating both cancer staging and treatment monitoring to more rapidly identify an effective therapeutic approach.Here, we evaluated 2 cystine knot peptides, R01 and S02, previously engineered with a 3-6 nM affinity for integrin ?v?6, 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 ?v?6-positive) or 293 (integrin ?v?6-negative) tumors. Small-animal PET scans were acquired at 0.5, 1, and 2 h after injection. Ex vivo ?-counting of dissected tissues was performed at 0.5 and 2 h.(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 ?v?6-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 ?-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).These cystine knot peptide tracers, in particular (18)F-fluorobenzoate-R01, show translational promise for molecular imaging of integrin ?v?6 overexpression in pancreatic and other cancers.
| S-EPMC4130341 | biostudies-literature
Project description:BackgroundCystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor.Methodology/principal findingsWe used a rational loop-grafting approach to engineer AgTx variants that bound to ?v?3 integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants. Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI. Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins.Conclusions/significanceIn this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.
| S-EPMC3616073 | biostudies-literature
Project description:Twenty million Americans suffer from peripheral nerve injury caused by trauma and medical disorders, resulting in a broad spectrum of potentially debilitating side effects. In one out of four cases, patients identify surgery as the root cause of their nerve injury. Particularly during tumor resections or after traumatic injuries, tissue distortion and poor visibility can challenge a surgeon's ability to precisely locate and preserve peripheral nerves. Intuitively, surgical outcomes would improve tremendously if nerves could be highlighted using an exogeneous contrast agent. In clinical practice, however, the current standard of care-visual examination and palpation-remains unchanged. To address this unmet clinical need, we explored the expression of voltage-gated sodium channel Nav1.7 as an intraoperative marker for the peripheral nervous system. We show that expression of Nav1.7 is high in peripheral nerves harvested from both human and mouse tissue. We further show that modification of a Nav1.7-selective peptide, Hsp1a, can serve as a targeted vector for delivering a fluorescent sensor to the peripheral nervous system. Ex vivo, we observe a high signal-to-noise ratio for fluorescently labeled Hsp1a in both histologically prepared and fresh tissue. Using a surgical fluorescent microscope, we show in a simulated clinical scenario that the identification of mouse sciatic nerves is possible, suggesting that fluorescently labeled Hsp1a tracers could be used to discriminate nerves from their surrounding tissues in a routine clinical setting.
| S-EPMC7372312 | biostudies-literature
Project description:PurposeHepatocellular carcinoma (HCC) remains one of the most challenging diseases worldwide. Glypican-3 (GPC-3) is a cell surface proteoglycan that is overexpressed on the membrane of HCC cells. The purpose of this study was to develop a target-specific radiofluorinated peptide for positron emission tomography (PET) imaging of GPC3 expression in hepatocellular carcinoma.ProceduresNew GPC3-binding peptides (GP2076 and GP2633) were radiolabeled with F-18 using Al[18F]F labeling approach, and the resulting PET probes were subsequently subject to biological evaluations. A highly hydrophilic linker was incorporated into GP2633 with an aim of reducing the probe uptake in liver and increasing tumor-to-liver (T/L) contrast. Both GP2076 and GP2633 were radiolabeled using Al[18F]F chelation approach. The binding affinity, octanol/water partition coefficient, cellular uptake and efflux, and stability of both F-18 labeled peptides were tested. Tumor targeting efficacy and biodistribution of Al[18F]F-GP2076 and Al[18F]F-GP2633 were determined by PET imaging in HCC-bearing mice. Immunohistochemistry analyses were performed to compare the findings from PET scans.ResultsAl[18F]F-GP2076 and Al[18F]F-GP2633 were rapidly radiosynthesized within 20 min in excellent radiochemical purity (>?97 %). Al[18F]F-GP2633 was determined to be more hydrophilic than Al[18F]F-GP2076 in terms of octanol/water partition coefficient. Both Al[18F]F-GP2076 and Al[18F]F-GP2633 demonstrated good in vitro and in vivo stability and binding specificity to GPC3-positive HepG2 cells. For PET imaging, Al[18F]F-GP2633 exhibited enhanced uptake in HepG2 tumor (%ID/g 3.37?±?0.35 vs. 2.13?±?0.55, P?=?0.031) and reduced accumulation in liver (%ID/g 1.70?±?0.26 vs. 3.70?±?0.98, P?=?0.027) at 60 min post-injection (pi) as compared to Al[18F]F-GP2076, resulting in significantly improved tumor-to-liver (T/L) contrast (ratio 2.00?±?0.18 vs. 0.59?±?0.14, P?=?0.0004). Higher uptake of Al[18F]F-GP2633 in GPC3-positive HepG2 tumor was observed as compared to GPC3-negative McA-RH7777 tumor (%ID/g 3.37?±?0.35 vs. 1.64?±?0.03, P?=?0.001) at 60 min pi, confirming GPC3-specific accumulation of Al[18F]F-GP2633 in HepG2 tumor.ConclusionThe results demonstrated that Al[18F]F-GP2633 is a promising probe for PET imaging of GPC3 expression in HCC. Convenient preparation, excellent GPC3 specificity in HCC, and favorable excretion profile of Al[18F]F-GP2633 warrant further investigation for clinical translation. PET imaging with a GPC3-specific probe would provide clinicians with vital diagnostic information that could have a significant impact on the management of HCC patients.
| S-EPMC7007182 | biostudies-literature
Project description:Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide ?-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native ?-PVIIA, cyclic ?-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated ?-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of ?-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules. Biotechnol. Bioeng. 2016;113: 2202-2212. © 2016 Wiley Periodicals, Inc.
| S-EPMC5526200 | biostudies-literature
Project description:The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications.
| S-EPMC6962393 | biostudies-literature
Project description: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 αvβ6 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 αvβ6 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 αvβ6-positive tumors.MethodsAn engineered cystine knot peptide, R01, that recognizes integrin αvβ6 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 αvβ6-positive) or 293T (integrin αvβ6-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.ResultsThe A740-R01 demonstrated integrin αvβ6-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.ConclusionA740-R01 specifically targeted integrin αvβ6 with low nanomolar affinity. A740-R01 was able to detect integrin αvβ6 both in vitro and in vivo by photoacoustic and fluorescence imaging. A740-R01 is able to detect αvβ6-positive tumors in living subjects and may have clinical application in cancer diagnosis and real-time image-guided surgery.
| S-EPMC5367447 | biostudies-literature
Project description:UnlabelledRecently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to alpha(v)beta(3) integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects.MethodsEngineered AgRP peptides were prepared by solid-phase peptide synthesis and were folded in vitro and purified by reversed-phase high-performance liquid chromatography. Competition assays were used to measure the relative binding affinities of engineered AgRP peptides for integrin receptors expressed on the surface of U87MG glioblastoma cells. The highest-affinity mutant, AgRP clone 7C, was site-specifically conjugated with 1,4,7,10-tetra-azacyclododecane-N,N',N''N'''-tetraacetic acid (DOTA). The resulting bioconjugate, DOTA-AgRP-7C, was radiolabeled with (64)Cu for biodistribution analysis and small-animal PET studies in mice bearing U87MG tumor xenografts. In addition to serum stability, the in vivo metabolic stability of (64)Cu-DOTA-AgRP-7C was assessed after injection and probe recovery from mouse kidney, liver, tumor, and urine.ResultsAgRP-7C and DOTA-AgRP-7C bound with high affinity to integrin receptors expressed on U87MG cells (half maximal inhibitory concentration values, 20 +/- 4 and 14 +/- 2 nM, respectively). DOTA-AgRP-7C was labeled with (64)Cu with high radiochemical purity (>99%). In biodistribution and small-animal PET studies, (64)Cu-DOTA-AgRP-7C displayed rapid blood clearance, good tumor uptake and retention (2.70 +/- 0.93 percentage injected dose per gram [%ID/g] and 2.37 +/- 1.04 %ID/g at 2 and 24 h, respectively), and high tumor-to-background tissue ratios. The integrin-binding specificity of (64)Cu-DOTA-AgRP-7C was confirmed in vitro and in vivo by showing that a large molar excess of the unlabeled peptidomimetic c(RGDyK) could block probe binding and tumor uptake. Serum stability and in vivo metabolite assays demonstrated that engineered AgRP peptides are sufficiently stable for in vivo molecular imaging applications.ConclusionA radiolabeled version of the engineered AgRP peptide 7C showed promise as a PET agent for tumors that express the alpha(v)beta(3) integrin. Collectively, these results validate AgRP-based cystine-knot peptides for use in vivo as molecular imaging agents and provide support for the general use of AgRP as a scaffold to develop targeting peptides, and hence diagnostics, against other tumor receptors.
| S-EPMC4143171 | biostudies-literature
Project description:A novel cystine knot peptide, asteropsin E (ASPE), was isolated from an Asteropus sp. marine sponge. The primary, secondary, and tertiary structures of ASPE were determined by high-resolution 2D NMR spectroscopy (900 MHz). With the exception of an N-terminal modification, ASPE shares properties with the previously reported asteropsins A-D, that is, the absence of basic residues, a highly acidic nature, conserved structurally important residues (including two cis-prolines), and a highly conserved tertiary structural framework. ASPE was found to be remarkably stable to gastrointestinal tract enzymes (chymotrypsin, elastase, pepsin, and trypsin) and to human plasma.
| S-EPMC4128683 | biostudies-literature