Optimal His-Tag Design for Efficient [99mTc(CO)3]+ and [188Re(CO)3]+ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [99mTc(CO)3]+ and [188Re(CO)3]+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [99mTc(CO)3]+ and [188Re(CO)3]+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [99mTc(CO)3]+ or [188Re(CO)3]+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of [Re(CO)3(H2O)3]+ and electrophoresis of solutions of [99mTc(CO)3(H2O)3]+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

A one-pot three-component radiochemical reaction for rapid assembly of 125I-labeled molecular probes.

Nuclear imaging in conjunction with radioactive tracers enables noninvasive measurements of biochemical events in vivo. However, access to tracers remains limited due to the lack of methods for rapid assembly of radiolabeled molecules with the prerequisite biological activity. Herein, we report a one-pot, three-component, copper(II)-mediated reaction of azides, alkynes, and [(125)I]iodide to yield 5-[(125)I]iodo-1,2,3-triazoles. Using a selection of azides and alkynes in a combinatorial approach, we have synthesized a library of structurally diverse (125)I-labeled triazoles functionalized with bioconjugation groups, fluorescent dyes, and biomolecules. Our preliminary biological evaluation suggests that 5-[(125)I]iodo-1,2,3-triazoles are resistant to deiodination in vivo, both as small molecular probes and as antibody conjugates. The ability to incorporate radioactive iodide into triazoles directly from the parent azides and alkynes makes the method broadly applicable and offers the potential to rapidly assemble molecular probes from an array of structurally diverse, and readily available, building blocks.

Evaluation of a 125I-labelled benzazepinone derived voltage-gated sodium channel blocker for imaging with SPECT.

Voltage-gated sodium channels (VGSCs) are a family of transmembrane proteins that mediate fast neurotransmission, and are integral to sustain physiological conditions and higher cognitive functions. Imaging of VGSCs in vivo holds promise as a tool to elucidate operational functions in the brain and to aid the treatment of a wide range of neurological diseases. To assess the suitability of 1-benzazepin-2-one derived VGSC blockers for imaging, we have prepared a (125)I-labelled analogue of BNZA and evaluated the tracer in vivo. In an automated patch-clamp assay, a diastereomeric mixture of the non-radioactive compound blocked the Na(v)1.2 and Na(v)1.7 VGSC isoforms with IC(50) values of 4.1 ± 1.5 µM and 0.25 ± 0.07 µM, respectively. [(3)H]BTX displacement studies revealed a three-fold difference in affinity between the two diastereomers. Iodo-destannylation of a tin precursor with iodine-125 afforded the two diastereomerically pure tracers, which were used to assess binding to VGSCs in vivo by comparing their tissue distributions in mice. Whilst the results point to a lack of VGSC binding in vivo, SPECT imaging revealed highly localized uptake in the interscapular region, an area typically associated with brown adipose tissue, which in addition to high metabolic stability of the iodinated tracer, demonstrate the potential of 1-benzazepin-2-ones for in vivo imaging.

Author Correction: Non-Invasive whole-body detection of complement activation using radionuclide imaging in a mouse model of myocardial ischaemia-reperfusion injury.

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Non-Invasive whole-body detection of complement activation using radionuclide imaging in a mouse model of myocardial ischaemia-reperfusion injury.

Complement activation is a recognised mediator of myocardial ischaemia-reperfusion-injury (IRI) and cardiomyocytes are a known source of complement proteins including the central component C3, whose activation products can mediate tissue inflammation, cell death and profibrotic signalling. We investigated the potential to detect and quantify the stable covalently bound product C3d by external body imaging, as a marker of complement activation in heart muscle in a murine model of myocardial IRI. We used single-photon-emission-computed-tomography (SPECT) in conjunction with 99mTechnecium-labelled recombinant complement receptor 2 (99mTc-rCR2), which specifically detects C3d at the site of complement activation. Compared to control imaging with an inactive CR2 mutant (99mTc-K41E CR2) or an irrelevant protein (99mTc-PSMA) or using 99mTc-rCR2 in C3-deficient mice, the use of 99mTc-rCR2 in complement-intact mice gave specific uptake in the reperfused myocardium. The heart to skeletal muscle ratio of 99mTc-rCR2 was significantly higher than in the three control groups. Histological analysis confirmed specific uptake of 99mTc-rCR2. Following therapeutic inhibition of complement C3 activation, we found reduced myocardial uptake of 99mTc-rCR2. We conclude, therefore that 99mTc-rCR2 imaging can be used for non-invasive detection of activated complement and in future could be exploited to quantify the severity of myocardial damage due to complement activation.

Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans.

Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.

Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.

The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins.

Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging.

BACKGROUND: In vivo imaging using genetic reporters is a central supporting tool in the development of cell and gene therapies affording us the ability to selectively track the therapeutic indefinitely. Previous studies have demonstrated the utility of the human norepinephrine transporter (hNET) as a positron emission tomography/single photon emission computed tomography (PET/SPECT) genetic reporter for in vivo cellular imaging. Here, our aim was to extend on this work and construct a tricistronic vector with dual optical (firefly luciferase) and nuclear (hNET) in vivo imaging and ex vivo histochemical capabilities. Guiding this development, we describe how a fluorescent substrate for hNET, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)), can be used to optimise vector design and serve as an in vitro functional screen. METHODS: Vectors were designed to co-express a bright red-shifted firefly luciferase (FLuc), hNET and a small marker gene RQR8. Genes were co-expressed using 2A peptide linkage, and vectors were transduced into a T cell line, SupT1. Two vectors were constructed with different gene orientations; FLuc.2A.RQR8.2A.hNET and hNET.2A.FLuc.2A.RQR8. hNET function was assessed using ASP(+)-guided flow cytometry. In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice. RESULTS: SupT1/FLuc.2A.RQR8.2A.hNET cells resulted in >4-fold higher ASP(+) uptake compared to SupT1/hNET.2A.FLuc.2A.RQR8, suggesting that 2A orientation effected hNET function. SupT1/FLuc.2A.RQR8.2A.hNET cells were readily visualised with both BLI and SPECT, demonstrating high signal to noise at 24 h post (123)I-meta-iodobenzylguanidine (MIBG) administration. CONCLUSIONS: In this study, a pre-clinical tricistronic vector with flow cytometry, BLI, SPECT and histochemical capabilities was constructed, which can be widely applied in cell tracking studies supporting the development of cell therapies. The study further demonstrates that hNET function in engineered cells can be assessed using ASP(+)-guided flow cytometry in place of costly radiosubstrate methodologies. This fluorogenic approach is unique to the hNET PET/SPECT reporter and may prove valuable when screening large numbers of cell lines or vector/mutant constructs.

Significant Therapeutic Efficacy with Combined Radioimmunotherapy and Cetuximab in Preclinical Models of Colorectal Cancer.

UNLABELLED: Despite extensive efforts to improve the clinical management of patients with colorectal cancer, approved treatments for advanced disease offer limited survival benefit. Therefore, the identification of novel treatment strategies is essential. We evaluated the preclinical efficacy of combination radioimmunotherapy, using a humanized (131)I-labeled anti-carcinoembryonic antigen antibody ((131)I-huA5B7), with cetuximab in colorectal cancer (CRC). METHODS: Three human CRC cell lines--SW1222, LoVo, and LS174T--were used to generate subcutaneous xenografts, and stably luciferase-transfected SW1222 cells were used to establish a model of hepatic metastases in immunocompromised mice. Imaging and biodistribution studies were conducted to confirm the selective tumor localization of (131)I-huA5B7. Efficacy was evaluated on the basis of tumor growth delay and survival, along with markers of DNA damage response, cell cycle, proliferation, and apoptosis. RESULTS: Selective tumor targeting was achieved with (131)I-huA5B7 alone or in combination with cetuximab without observable toxicity. Compared with monotherapy, combining cetuximab with radioimmunotherapy significantly and synergistically reduced tumor growth and prolonged survival of mice in 2 of the subcutaneous and in the metastatic tumor model. Evidence of DNA damage, G2/M arrest, significantly decreased proliferation, and increased apoptosis were observed with radioimmunotherapy and the combination therapy. However, a significant decrease in DNA-protein kinase expression with the combination regimen suggests that the addition of cetuximab suppressed DNA repair. CONCLUSION: Our results demonstrate enhanced therapeutic efficacy with the combination of cetuximab and radioimmunotherapy in CRC, which could potentially translate into successful clinical outcomes. This strategy could improve the treatment of residual disease postoperatively and ultimately prevent or delay recurrence. Furthermore, other carcinoembryonic antigen-expressing malignancies could also benefit from this approach.

Preferential targeting of disseminated liver tumors using a recombinant adeno-associated viral vector.

A novel selectively targeting gene delivery approach has been developed for advanced hepatocellular carcinoma (HCC), a leading cause of cancer mortality whose prognosis remains poor. We combine the strong liver tropism of serotype-8 capsid-pseudotyped adeno-associated viral vectors (AAV8) with a liver-specific promoter (HLP) and microRNA-122a (miR-122a)-mediated posttranscriptional regulation. Systemic administration of our AAV8 construct resulted in preferential transduction of the liver and encouragingly of HCC at heterotopic sites, a finding that could be exploited to target disseminated disease. Tumor selectivity was enhanced by inclusion of miR-122a-binding sequences (ssAAV8-HLP-TK-122aT4) in the expression cassette, resulting in abrogation of transgene expression in normal murine liver but not in HCC. Systemic administration of our tumor-selective vector encoding herpes simplex virus-thymidine kinase (TK) suicide gene resulted in a sevenfold reduction in HCC growth in a syngeneic murine model without toxicity. In summary, we have developed a systemically deliverable gene transfer approach that enables high-level expression of therapeutic genes in HCC but not normal tissues, thus improving the prospects of safe and effective treatment for advanced HCC.

Development of Fluorine-18 Labeled Metabolically Activated Tracers for Imaging of Drug Efflux Transporters with Positron Emission Tomography.

Increased activity of efflux transporters, e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier is a pathological hallmark of many neurological diseases, and the resulting multiple drug resistance represents a major clinical challenge. Noninvasive imaging of transporter activity can help to clarify the underlying mechanisms of drug resistance and facilitate diagnosis, patient stratification, and treatment monitoring. We have developed a metabolically activated radiotracer for functional imaging of P-gp/BCRP activity with positron emission tomography (PET). In preclinical studies, the tracer showed excellent initial brain uptake and clean conversion to the desired metabolite, although at a sluggish rate. Blocking with P-gp/BCRP modulators led to increased levels of brain radioactivity; however, dynamic PET did not show differential clearance rates between treatment and control groups. Our results provide proof-of-concept for development of prodrug tracers for imaging of P-gp/BCRP function in vivo but also highlight some challenges associated with this strategy.

Optimising the radiolabelling properties of technetium tricarbonyl and His-tagged proteins.

BACKGROUND: To date, the majority of protein-based radiopharmaceuticals have been radiolabelled using non-site-specific conjugation methods, with little or no control to ensure retained protein function post-labelling. The incorporation of a hexahistidine sequence (His-tag) in a recombinant protein can be used to site-specifically radiolabel with 99mTc-tricarbonyl ([99mTc(CO)3]+). This chemistry has been made accessible via a technetium tricarbonyl kit; however, reports of radiolabelling efficiencies and specific activities have varied greatly from one protein to another. Here, we aim to optimise the technetium tricarbonyl radiolabelling method to produce consistently >95% radiolabelling efficiencies with high specific activities suitable for in vivo imaging. METHODS: Four different recombinant His-tagged proteins (recombinant complement receptor 2 (rCR2) and three single chain antibodies, α-CD33 scFv, α-VCAM-1 scFv and α-PSMA scFv), were used to study the effect of kit volume, ionic strength, pH and temperature on radiolabelling of four proteins. RESULTS: We used 260 and 350 µL [99mTc(CO)3]+ kits enabling us to radiolabel at higher [99mTc(CO)3]+ and protein concentrations in a smaller volume and thus increase the rate at which maximum labelling efficiency and specific activity were reached. We also demonstrated that increasing the ionic strength of the reaction medium by increasing [Na+] from 0.25 to 0.63 M significantly increases the rate at which all four proteins reach a >95% labelling efficiency by at least fourfold, as compared to the conventional IsoLink® kit (Covidien, Petten, The Netherlands) and 0.25 M [Na+]. CONCLUSION: We have found optimised kit and protein radiolabelling conditions suitable for the reproducible, fast, efficient radiolabelling of proteins without the need for post-labelling purification.

Development of purine-derived 18F-labeled pro-drug tracers for imaging of MRP1 activity with PET.

Multidrug resistance-associated protein 1 (MRP1) is a drug efflux transporter that has been implicated in the pathology of several neurological diseases and is associated with development of multidrug resistance. To enable measurement of MRP1 function in the living brain, a series of 6-halopurines decorated with fluorinated side chains have been synthesized and evaluated as putative pro-drug tracers. The tracers were designed to undergo conjugation with glutathione within the brain and hence form the corresponding MRP1 substrate tracers in situ. 6-Bromo-7-(2-[(18)F]fluoroethyl)purine showed good brain uptake and rapid metabolic conversion. Dynamic PET imaging demonstrated a marked difference in brain clearance rates between wild-type and mrp1 knockout mice, suggesting that the tracer can allow noninvasive assessment of MRP1 activity in vivo.

Incorporation of paramagnetic, fluorescent and PET/SPECT contrast agents into liposomes for multimodal imaging.

A series of metal-chelating lipid conjugates has been designed and synthesized. Each member of the series bears a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) macrocycle attached to the lipid head group, using short n-ethylene glycol (n-EG) spacers of varying length. Liposomes incorporating these lipids, chelated to Gd(3+), (64)Cu(2+), or (111)In(3+), and also incorporating fluorescent lipids, have been prepared, and their application in optical, magnetic resonance (MR) and single-photon emission tomography (SPECT) imaging of cellular uptake and distribution investigated in vitro and in vivo. We have shown that these multimodal liposomes can be used as functional MR contrast agents as well as radionuclide tracers for SPECT, and that they can be optimized for each application. When shielded liposomes were formulated incorporating 50% of a lipid with a short n-EG spacer, to give nanoparticles with a shallow but even coverage of n-EG, they showed good cellular internalization in a range of tumour cells, compared to the limited cellular uptake of conventional shielded liposomes formulated with 7% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)(2000)] (DSPE-PEG2000). Moreover, by matching the depth of n-EG coverage to the length of the n-EG spacers of the DOTA lipids, we have shown that similar distributions and blood half lives to DSPE-PEG2000-stabilized liposomes can be achieved. The ability to tune the imaging properties and distribution of these liposomes allows for the future development of a flexible tri-modal imaging agent.

Recombinant complement receptor 2 radiolabeled with [99mTc(CO)3]+: a potential new radiopharmaceutical for imaging activated complement.

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [(99m)Tc(CO)(3)(OH(2))(3)](+)). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d(+) red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d(+), in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a K(D)≈500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [(99m)Tc(CO)(3)(OH(2))(3)](+) at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d(+) RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d(+) RBCs. We conclude that rCR2-Tc(99m) has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent.