Q2DSTD NMR deciphers epitope-mapping variability for peptide recognition of integrin ?v?6.
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ABSTRACT:
SUBMITTER: Sorge JL
PROVIDER: S-EPMC4541471 | biostudies-literature | 2015 Aug
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.
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Project description:The epithelial integrin αvβ6 is expressed by many malignant carcinoma cell types, including pancreatic cancer, and thus represents a promising target for radionuclide therapy. The peptide cyclo(FRGDLAFp(NMe)K) was decorated with different chelators (DOTPI, DOTAGA, and DOTA). The Lu(III) complexes of these conjugates exhibited comparable αvβ6 integrin affinities (IC50 ranging from 0.3 to 0.8 nM) and good selectivities against other integrins (IC50 for αvβ8 >43 nM; for α5β1 >238 nM; and for αvβ3, αvβ5, and αIIbβ3 >1000 nM). Although different formal charges of the Lu(III) chelates (ranging from 0 to 4) resulted in strongly varying degrees of hydrophilicity (log D ranging from -3.0 to -4.1), biodistributions in murine H2009 xenografts of the Lu-177-labeled compounds (except the DOTPI derivative) were quite similar and comparable to our previously reported αvβ6 integrin positron emission tomography tracer Ga-68-avebehexin. Hence, combinations of existing Ga-68- and Lu-177-labeled c(FRGDLAFp(NMe)K) derivatives could be utilized for αvβ6 integrin-targeted theranostics, whereas our data nonetheless suggest that further improvement of pharmacokinetics might be necessary to ensure clinical success.
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Project description:The integrin αvβ6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvβ6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvβ6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvβ6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvβ6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvβ6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvβ6 in vivo, expanding opportunities for cancer targeting and therapy.
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Project description:Multimerization is an established strategy to design bioactive macromolecules with enhanced avidity, which has been widely employed to increase the target-specific binding and uptake of imaging probes and pharmaceuticals. However, the factors governing the general biodistribution of multimeric probes are less well understood but are nonetheless decisive for their clinical application. We found that regiospecific exchange of phenylalanine by tyrosine (chemically equivalent to addition of single oxygen atoms) can have an unexpected, dramatic impact on the in vivo behavior of gallium-68 labeled αvβ6-integrin binding peptides trimers. For example, introduction of one and two Tyr, equivalent to just 1 and 2 additional oxygens and molecular weight increases of 0.38% and 0.76% for our >4 kDa constructs, reduced non-specific liver uptake by 50% and 72%, respectively. The observed effect did not correlate to established polarity measures such as log D, and generally defies explanation by reductionist approaches. We conclude that multimers should be viewed not just as molecular combinations of peptides whose properties simply add up, but as whole entities with higher intrinsic complexity and thus a strong tendency to exhibit newly emerged properties that, on principle, cannot be predicted from the characteristics of the monomers used.
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Project description:Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.
| S-EPMC5529020 | biostudies-literature
Project description:Exosomes, cell-derived vesicles of endosomal origin, are continuously released in the extracellular environment and play a key role in intercellular crosstalk. In this study, we have investigated whether transfer of integrins through exosomes between prostate cancer (PrCa) cells occurs and whether transferred integrins promote cell adhesion and migration. Among others, we have focused on the αvβ6 integrin, which is not detectable in normal human prostate but is highly expressed in human primary PrCa as well as murine PrCa in Pten(pc-/-) mice. After confirming the fidelity of the exosome preparations by electron microscopy, density gradient, and immunoblotting, we determined that the αvβ6 integrin is actively packaged into exosomes isolated from PC3 and RWPE PrCa cell lines. We also demonstrate that αvβ6 is efficiently transferred via exosomes from a donor cell to an αvβ6-negative recipient cell and localizes to the cell surface. De novo αvβ6 expression in an αvβ6-negative recipient cell is not a result of a change in mRNA levels but is a consequence of exosome-mediated transfer of this integrin between different PrCa cells. Recipient cells incubated with exosomes containing αvβ6 migrate on an αvβ6 specific substrate, latency-associated peptide-TGFβ, to a greater extent than cells treated with exosomes in which αvβ6 is stably or transiently down-regulated by shRNA or siRNA, respectively. Overall, this study shows that exosomes from PrCa cells may contribute to a horizontal propagation of integrin-associated phenotypes, which would promote cell migration, and consequently, metastasis in a paracrine fashion.
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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.
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Project description:Encouraging results from recent clinical trials are revitalizing the field of oncolytic virotherapies. Human adenovirus type 5 (HAdV-C5/Ad5) is a common vector for its ease of manipulation, high production titers and capacity to transduce multiple cell types. However, effective clinical applications are hindered by poor tumor-selectivity and vector neutralization. We generated Ad5/kn48 by pseudotyping Ad5 with the fiber knob domain from the less seroprevalent HAdV-D48 (Ad48). The vector was shown to utilize coxsackie and adenovirus receptor (CAR) but not CD46 for cell entry. A 20-amino acid peptide NAVPNLRGDLQVLAQKVART (A20) was inserted into the Ad5. Luc HI loop (Ad5.HI.A20) and Ad5/kn48 DG loop (Ad5/kn48.DG.A20) to target a prognostic cancer cell marker, αvβ6 integrin. Relative to the Ad5.Luc parent vector, Ad5.HI.A20, Ad5.KO1.HI.A20 (KO1, ablated CAR-binding) and Ad5/kn48.DG.A20 showed ~ 160-, 270- and 180-fold increased transduction in BT-20 breast carcinoma cells (αvβ6high). Primary human epithelial ovarian cancer (EOC) cultures derived from clinical ascites provided a useful ex vivo model for intraperitoneal virotherapy. Ad5.HI.A20, Ad5.KO1.HI.A20 and Ad5/kn48.DG.A20 transduction was ~ 70-, 60- and 16-fold increased relative to Ad5.Luc in EOC cells (αvβ6high), respectively. A20 vectors transduced EOC cells at up to ~ 950-fold higher efficiency in the presence of neutralizing ovarian ascites, as compared to Ad5.Luc. Efficient transduction and enhanced cancer-selectivity via a non-native αvβ6-mediated route was demonstrated, even in the presence of pre-existing anti-Ad5 immunity. Consequently, αvβ6-targeted Ad vectors may represent a promising platform for local intraperitoneal treatment of ovarian cancer metastases.
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Project description:BackgroundPatients with primary sclerosing cholangitis (PSC) possess autoantibodies against biliary epithelial cells. However, the target molecules remain unknown.MethodsThe sera of patients with PSC and controls were subjected to enzyme-linked immunosorbent assays to detect autoantibodies using recombinant integrin proteins. Integrin αvβ6 expression in the bile duct tissues was examined using immunofluorescence. The blocking activity of the autoantibodies was examined using solid-phase binding assays.ResultsAnti-integrin αvβ6 antibodies were detected in 49/55 (89.1%) patients with PSC and 5/150 (3.3%) controls (P < 0.001), with a sensitivity and specificity of 89.1% and 96.7%, respectively, for PSC diagnosis. When focusing on the presence or absence of IBD, the proportion of the positive antibodies in PSC with IBD was 97.2% (35/36) and that in PSC alone was 73.7% (14/19) (P = 0.008). Integrin αvβ6 was expressed in bile duct epithelial cells. Immunoglobulin (Ig)G from 15/33 patients with PSC blocked integrin αvβ6-fibronectin binding through an RGD (Arg-Gly-Asp) tripeptide motif.ConclusionsAutoantibodies against integrin αvβ6 were detected in most patients with PSC; anti-integrin αvβ6 antibody may serve as a potential diagnostic biomarker for PSC.
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Project description:Alzheimer´s Disease (AD) is one of the most common neurodegenerative disorders worldwide. Excess of ?-amyloid (A?), a peptide with a high propensity to misfold and self-aggregate, is believed to be the major contributor to the observed neuronal degeneration and cognitive decline in AD. Here, we characterize the epitope of a novel anti-A? monoclonal antibody, the STAB-MAb, which has previously demonstrated picomolar affinities for both monomers (KD?=?80 pM) and fibrils (KD?=?130 pM) of A?(1-42) and has shown therapeutic efficacy in preclinical mouse models of AD. Our findings reveal a widespread epitope that embraces several key A? residues that have been previously described as important in the A? fibrillation process. Of note, STAB-MAb exhibits a stronger affinity for the N-terminus of A? and stabilizes an ?-helix conformation in the central to N-terminal region of the peptide, in addition to disrupting a characteristic salt-bridge of a hairpin structure present in fibrils. The NMR derived epitope supports the observed results from ThT-monitored fluorescence and electron microscopy experiments, in which STAB-MAb was shown to inhibit the formation of aggregates and promote disruption of pre-formed fibrils. In combination with the published in vitro and in vivo assays, our study highlights STAB-MAb as a rare and versatile antibody with analytical, diagnostic and therapeutic efficacy.
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