Optimising the radiolabelling properties of technetium tricarbonyl and His-tagged proteins
- Adam Badar†1, 2,
- Jennifer Williams†1,
- Rafael TM de Rosales1,
- Richard Tavaré1, 3,
- Florian Kampmeier1,
- Philip J Blower1 and
- Gregory ED Mullen1Email author
© Badar et al.; licensee Springer. 2014
Received: 31 January 2014
Accepted: 21 February 2014
Published: 7 March 2014
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.
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.
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+].
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.
KeywordsProtein radiolabelling Site-specific radiolabelling 99mTc tricarbonyl His-tag radiolabelling Technetium-99 m
The specificity and affinity of recombinant proteins and antibodies targeted towards antigens make them highly attractive as a basis for radiopharmaceuticals for molecular imaging. To retain these key attributes, it is essential not to compromise the recognition function of the protein when radiolabelling. This can be achieved using site-specific labelling methods that exert maximum control over the number and site of modification(s) to the molecule, while still maintaining protein function. Such methodologies should give rise to homogeneous conjugates with reproducible chemical and pharmacological properties. They must ensure that the conjugation of the radiochelate is outside of the target binding site or at a distinct site known not to affect antigen binding. Ideally, radiolabelling would be achieved rapidly, under mild conditions, to a high specific activity and preferably in a simple one-pot kit-based method without the need for subsequent purification steps.
Waibel et al.  developed an elegant method that permits radiolabelling of proteins engineered with sequences of additional histidine residues known as His-tags. The His-tag was originally developed to facilitate purification of recombinant proteins using metal chelate-based affinity chromatography. Radiolabelling of His-tags could be achieved with technetium-99 m (99mTc) in its stable + I oxidation state in the form of 99mTc-tricarbonyl ([99mTc(CO)3]+). This organometallic complex is produced in the form of its aqua ion [99mTc(CO)3(H2O)3]+ in a one-step reaction by reduction of the generator-eluted form of 99mTc, sodium pertechnetate ([99mTcO4]-). Histidine has been demonstrated to be the favoured [99mTc(CO)3]+-binding ligand among amino acids and labelling efficiency and stability increase with increased number of engineered histidines . Direct labelling of non-His-tag proteins with [99mTc(CO)3]+ has previously resulted in poor stability and low labelling efficiency and specific activity, indicating that other potential amino acid side chains donor groups such as thiol, thioether, carboxylate and amine do not make a significant contribution in the absence of histidines [3–9]. Modification of the His-tag sequence from HHHHHH to HEHEHE in order to improve tracer biodistribution also resulted in reduced labelling efficiency [10, 11]. In contrast, by engineering an additional cysteine seven amino acids downstream of the His-tag, Tavaré et al. demonstrated an improvement in labelling efficiency and specific activity compared to His-tag alone  and showed for the first time that the radiolabelling is indeed site-specific to the His-tagged region of the protein by carrying out tryptic digest and mass spectrometry on a His-tagged protein labelled with [Re(CO)3]+. The rhenium complex was only present in His-tag-containing fragments . Furthermore, we recently demonstrated that the engineered cysteine that increases radiolabelling efficiency is also involved in the coordination of the rhenium tricarbonyl . Several variants on labelling conditions have been studied to optimise the use of [99mTc(CO)3]+-labelling of His-tagged proteins, and there is a general agreement in the literature that labelling at neutral pH, high protein concentration and high temperature increase the rate of radiolabelling (see Additional file 1: Table S1) [1, 2, 12, 14–26]. These conditions, however, exclude proteins that are susceptible to aggregation or loss of function at high temperatures and concentrations or require particularly high specific activity [15, 23, 25]. To make this labelling chemistry readily accessible, a kit-based formulation has until recently been distributed by Mallinckrodt (subsidiary of Covidien, Petten, The Netherlands) under the trademarked name IsoLink. Since its introduction, IsoLink has supported the development of numerous protein-based imaging tracers. Despite the intent to provide a practical, simple and versatile radiolabelling method, there has been a large variation in labelling efficiencies reported in the literature, likely due to variations in protein/peptide properties and labelling conditions (see Additional file 1: Table S1), and no clinical trial has been performed to date with proteins labelled using the tricarbonyl labelling method.
The IsoLink kit comes in a 10-mL glass vial containing lyophilised reagents and is designed to reduce up to 3.7 GBq of [99mTcO4]- in a final volume of 1 mL. With the current composition, to achieve maximum specific activity (crucial for in vivo imaging), the protein must be added to this total volume, leading to low protein concentration and hence inefficient labelling  and high wastage. Recombinant proteins used in R&D are often precious, produced in low yield and available in small quantities and may be difficult to concentrate due to aggregation, precipitation or loss on columns or membranes. Furthermore, for preclinical work, volumes above 200 μL are not desirable or feasible for injection into mice (one tenth of total blood volume). The current kit necessitates an avoidable and time-consuming protein concentration step or leads to low specific activity. Thus, while labelling via the [99mTc(CO)3]+ method may be one of the most promising site-specific methods currently available for recombinant protein tracer development, the IsoLink kit in its current form is not optimal for routine use in preclinical research or for future clinical imaging. It needs to be optimised to achieve reproducible high labelling efficiencies (LE) and specific activities (SA) of recombinant proteins, without wasting large amounts of protein and 99mTc.
In this paper, we address how the kit could be further optimised to increase specific activity and radiolabelling efficiency of His-tagged proteins, at suitable labelling rates with reduced wastage of protein and radioactivity. We used four different His-tagged proteins to study the effect of various radiolabelling conditions.
IsoLink kits (generously provided by Covidien, Petten, The Netherlands) consisted of lyophilised formulation in an N2-flushed 10-mL glass vial, containing 8.5 mg sodium tartrate Na2C4H4O6, 2.85 mg sodium tetraborate Na2B4O7, 7.15 mg sodium carbonate Na2CO3 and 4.5 mg sodium boranocarbonate Na2H3BCO2. Kits are now available from an alternate supplier (Centre of Radiopharmaceutical Research at the Paul Scherrer Institute in Switzerland) or can be prepared according to Waibel et al. .
His-tagged proteins used in this study were (1) recombinant complement receptor 2 (rCR2) (16 kDa) recombinant protein , (2) α-CD33 scFv (29 kDa) , (3) α-PSMA scFv (27.7 kDa)  and (4) α-VCAM-1 scFv (28.8 kDa) . Protein concentration was measured by UV spectrometry using a Nanodrop device (ThermoScientific, Loughborough, Leicestershire, LE, UK) and a molar extinction coefficient of the respective protein determined by ProtParam .
Preparation of [99mTc(CO)3]+
Three methods were used and compared in the production of [99mTc(CO)3]+: a standard method employing an IsoLink kit according to the manufacturer's instructions (method 1), a method using the IsoLink kit with a reduced volume of [99mTcO4]- solution (method 2) and a new subdivided kit formulation (method 3).
Methods 1 and 2
Up to 2.5 GBq [99mTcO4]- in 1 mL (method 1) or in 350 to 400 μL (method 2) of saline was added to the kit and heated for 30 min at 100°C. The vial was then allowed to cool to room temperature (RT) and the solution neutralised with 145 to 160 μL (methods 1 and 2) of 1 M HCl to pH approximately 7.5 giving a total volume of 1,145 to 1,160 μL (method 1) or 495 to 560 μL (method 2), respectively.
IsoLink kits were reconstituted in 1 mL ultrapure milliQ water (Millipore, Billerica, MA, USA), degassed with nitrogen and subdivided into aliquots of 260 μL in microcentrifuge tubes with rubber ring-sealed screw caps (Nalgene, Rochester, NY, USA) under anaerobic environment. Aliquots were snap-frozen in liquid nitrogen, freeze-dried overnight, then stored at -80°C. Upon use, kits were allowed to thaw to RT, and up to 1 GBq [99mTcO4]-, generator elulate (100 μL) was added and heated for 30 min at 100°C. The microcentrifuge tube was then allowed to cool to RT and the solution neutralised with 25 μL of 1 M HCl to pH approximately 7.5 giving a total volume of 125 μL of [99mTc(CO)3]+.
To determine the radiochemical purity of the [99mTc(CO)3]+ produced, glass-backed silica gel 60 F254 thin layer chromatography (TLC) plates (3 cm × 7.5 cm, Merck™, Darmstadt, Germany) were used with a mobile phase of 1% HCl in methanol. Plates were analysed with a gamma ray TLC scanner (Lablogic, South Yorkshire, UK). Purity was expressed as a ratio of the [99mTc(CO)3]+ peak integral (Rf = 0.2 - 0.8) to integral of the whole chromatogram (including Rf = 0 for 99mTc colloids and Rf = 0.9 for unreduced [99mTcO4]-).
[99mTc(CO)3]+ radiolabelling of His-tagged recombinant proteins
To 100 μL of rCR2 (1 mg/mL, 62.5 μM) or α-CD33 scFv (1 mg/mL, 34.5 μM), or 20 μL of α-PSMA scFv (1 mg/mL, 36.1 μM) or α-VCAM-1 scFv (1 mg/mL, 34.7 μM) in PBS (0.14 M [Na+]), a 20-μL solution of [99mTc(CO)3]+ (prepared according to method 2 above for CD33 scFv, PSMA scFv and VCAM-1 scFv and method 3 for rCR2) was added. Each protein was radiolabelled in a final solution containing low, medium and high concentrations of Na+: 0.25 M [Na+] (low), 0.44 M [Na+] (medium) and 0.63 M [Na+] (high). The Na+ concentration of the [99mTc(CO)3]+ solution was adjusted with NaCl solution before being added to the protein. The concentration and volume of NaCl solution used was calculated according to the concentration of the Na+ required in the final protein-[99mTc(CO)3]+ solution after addition of [99mTc(CO)3]+ (see Additional file 1 for [Na+] calculations). Final protein concentrations were 0.85 mg/mL rCR2 (53.1 μM), 0.42 mg/mL α-CD33 scFv (14.5 μM) and α-PSMA scFv (15.2 μM), and 0.68 mg/mL α-VCAM-1 scFv (23.6 μM). The labelling reaction was carried out at 37°C and monitored for 2 h with labelling efficiency determined at 5, 15, 30, 60, 90, and 120 min using iTLC-SA paper with citrate buffer as the mobile phase. This system provided a definitive separation between the [99mTc(CO)3]+-labelled peptide (Rf = 0) and unbound [99mTc(CO)3]+ and unreduced 99mTcO4- (both with Rf = 1). The large separation between the peaks enabled the iTLC-SA strips to be cut in half and both the solvent front and baseline segments analysed using a gamma counter. Radiolabelling efficiency was expressed as a ratio of the [99mTc(CO)3]+ protein-labelled peak integral (Rf = 0) to the integral of the whole strip including unbound [99mTc(CO)3]+ and unconverted [99mTcO4]- (Rf = 1). The influence of pH, protein concentration and reaction temperature was evaluated using rCR2 and can be found in Additional file 1.
Determining site specificity with a His-tag challenge
A non-His-tagged protein (C2Ac ) was allowed to compete for radiolabel with a His-tagged protein (α-CD33 scFv) by incubating an equimolar mixture of both proteins (55 μM:55 μM) in a one-pot radiolabelling reaction using the IsoLink kit method 2 described above. As controls, the proteins were radiolabelled separately using 55 μM concentrations. The reactions were allowed to incubate for 1 h at 37°C, after which 5 μL samples were taken from each reaction mixture and loaded onto an SDS-PAGE gel (12% NuPage gel, Invitrogen, Paisley, PA, UK). After the gel was run, a reference lane with molecular weight markers was spotted with radioactivity at the 15- and 30-kDa bands. After protein separation via electrophoresis, phosphor screens (Perkin Elmer, Shelton, CT, USA) were exposed for 10 s, followed by imaging using a Cyclone® phosphorimager system (Perkin Elmer). The gel was analysed using ImageJ software (NIH, Bethesda, MD, USA), and the fraction of total radioactivity bound to each of C2Ac and α-CD33 scFv was determined via densitometry of the protein bands.
Reducing the volume of the IsoLink kit increases the [99mTc(CO)3]+ concentration while maintaining its radiochemical purity
High [Na+] improves specific activity and labelling efficiency when radiolabelling His-tagged proteins with [99mTc(CO)3]+
Time taken to reach 50% LE of four His-tagged proteins at varying [Na+]
0.25 M [Na+]
0.44 M [Na+]
0.63 M [Na+]
34 ± 1.4 min
10.2 ± 0.1 min
58.5 ± 6.3 min
31.7 ± 3.5 min
19.5 ± 2.4 min
15.2 ± 3.8 min
4.7 ± 0.8 min
3.9 ± 0.5 min
65.5 ± 10.5 min
19.9 ± 0.2 min
13.2 ± 2.6 min
Time taken to reach 95% LE of four His-tagged proteins at varying [Na+]
0.25 M [Na+]
0.44 M [Na+]
0.63 M [Na+]
71.1 ± 3.8 min
106.8 ± 2 min
68.9 ± 1.2 min
51.5 ± 14 min
34.6 ± 6.4 min
110.8 ± 5.1 min
73.2 ± 3.8 min
Non-specific binding of [99mTc(CO)3]+ does not occur in the presence of a His-tag
Technetium-99 m is well suited for small (<55 kDa) protein-based tracers (glomerular filtration molecular weight cutoff approximately 45 kDa below which the tracer has a markedly reduced blood clearance half-life). Its 6-h half-life is sufficiently long to carry out radiolabelling and allow for slower blood clearance of minutes to hours as compared to small molecules with seconds to minutes. Furthermore, 99mTc is readily available at low cost from transportable 99Mo/99mTc generators and has favourable photon energy for imaging while minimising patient radiation-absorbed dose. To radiolabel proteins with 99mTc, the tricarbonyl method developed by Waibel et al.  offers a convenient solution enabling labelling without prior modification with bifunctional chelators, under mild conditions in as little as 60 min, and has recently supported the development of new radiopharmaceuticals [12, 27].
A number of studies have demonstrated good stability of [99mTc(CO)3]+:His-tagged protein complex, with minimal transchelation or loss of bound activity, by challenging with high concentrations of histidine and cysteine or incubating in serum [12, 14, 16, 17]. Our group has recently shown that the complex specifically binds to the His-tag, demonstrated by the fact that rhenium tricarbonyl labelled His-tag protein post-tryptic digest is only bound to peptide fragments containing a His-tag . Previous reports have indicated that non-His-tag proteins can be radiolabelled with [99mTc(CO)3]+ albeit with relatively low efficiency, but little or no challenge or stability data was reported. By using a competitive binding assay, here we demonstrated that the technetium tricarbonyl indeed binds selectively to a His-tagged protein in the presence of a non-His-tagged protein and little or no (<2% ± 0.9%) non-specific labelling was observed.
Low radiolabelling efficiency (<95%) of radiopharmaceutical is problematic, with any unlabelled material competing for target, hampering quantitative measurements and imaging sensitivity, and requiring an additional purification step prior to imaging. High SA enables a radioactive signal using a low concentration of radiopharmaceutical well below the pharmacologically active dose and is crucial for imaging low-concentration targets. Radiolabelling the recombinant proteins according to the IsoLink instructions resulted in relatively poor SA and LE due to excessive dilution of the protein (Additional file 1: Figure S1).
As previously reported, we found that protein concentration, pH and temperature play a key role in achieving maximum SA and LE (Additional file 1: Figure S1) when labelling with technetium tricarbonyl. Achieving the optimal conditions however is currently hampered with the commercial IsoLink kit. To overcome this, we prepared modified kits that can produce a [99mTc(CO)3]+ solution with radiochemical purity equal to that of the unmodified kits but with higher radioactivity concentration. The lower volume means that the radiolabelled product can conveniently be used for preclinical in vivo studies, making more economical use of both protein and radioactivity. The higher protein and radionuclide concentration leads to a faster labelling reaction, in turn to achieving higher SA and LE in the same incubation time, or a shorter incubation time to reach the same SA and LE. Importantly, it reduces the incubation time, temperature and amount of protein needed to reach 95% LE (the threshold at which radiochemical purity is typically regarded as adequate for in vivo use) and hence makes it easier to avoid the need for post-labelling purification steps which can waste time and cause losses due to irreversible binding to columns and filters.
We also found that total [Na+] in the reaction mixture affects SA and LE, and this effect can be exploited by increasing [Na+] to increase the rate at which maximum SA and 95% LE are reached by up to fourfold. IsoLink kits contain a significant amount of Na+, 0.31 mmol, and the [Na+] of the final protein: [99mTc(CO)3]+ reaction mixture is dependent on the volume of the [99mTc(CO)3]+ solution added (see Additional file 1 for [Na+] calculations).
Using the conventional IsoLink kit, rates of labelling reactions and times to reach maximum SA and LE vary between proteins. These variations may be due to His-tag accessibility in the protein's tertiary structure or a charge effect from adjacent amino acids interacting with the positively charged tricarbonyl core. Increasing the ionic strength or [Na+] in the reaction mixture may contribute to a kinetically favourable binding environment. For example, at pH 7.5, one would expect one or more of the histidines in the His-tag to be positively charged and if, as reported in the literature, the technetium tricarbonyl precursor is also positively charged, the increase in the ionic strength of the buffer may dissipate the ionic repulsion between the species thus lowering the activation energy of binding.
In conclusion, the standard IsoLink kit, while reliably producing the technetium tricarbonyl precursor in high yield, is not optimal in scale for the routine and economic radiolabelling of different His-tag recombinant proteins at high specific activity for in vivo use. Each protein will need optimisation to achieve optimal SA and LE, and in some cases, adequate SA and LE may not be reached for routine clinical or preclinical use and purification steps are then required. By introducing modifications to the kit, it is possible to achieve reproducible and robust radiolabelling with optimal SA and LE in shorter incubation times and with smaller quantities of protein, avoid post-labelling purification steps and make the most efficient use of both protein and 99mTc, improving the prospects for clinical translation and commercialisation of the tricarbonyl kit.
We thank Hector Knight (Covidien) for providing the IsoLink kits, Dr Louise Emberson and Dr Peter Nicholls (University of Kent) for providing the α-CD33 scFv and Dr Jim Ballinger and the Nuclear Medicine Department of Guy’s and St Thomas’ NHS Foundation Trust for supplying 99mTc-pertechnetate. This research was supported by the Centre of Excellence in Medical Engineering Centre funded by the Wellcome Trust and EPSRC under grant number WT088641/Z/09/Z, the King's College London and UCL Comprehensive Cancer Imaging Centre funded by the CRUK and EPSRC in association with the MRC and DoH (England), and by the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.
- Waibel R, Alberto R, Willuda J, Finnern R, Schibli R, Stichelberger A, Egli A, Abram U, Mach JP, Plückthun A, Schubiger PA: Stable one-step technetium-99 m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex. Nat Biotechnol 1999, 17: 897–901. 10.1038/12890View ArticlePubMedGoogle Scholar
- Tait J, Smith C, Gibson D: Development of annexin V mutants suitable for labeling with Tc(I)-carbonyl complex. Bioconjug Chem 2002, 13: 1119–1123. 10.1021/bc025545sView ArticlePubMedGoogle Scholar
- Shah SQ, Khan AU, Khan MR: 99mTc(CO)3-Ibritumomab tiuxetan; a novel radioimmunoimaging (RII) agent of B cell non-Hodgkin’s lymphoma (NHL). Biol Chem 2012, 393: 71–75.View ArticlePubMedGoogle Scholar
- Chen WJ, Yen CL, Lo ST, Chen KT, Lo JM: Direct 99mTc labeling of Herceptin (trastuzumab) by 99mTc(I) tricarbonyl ion. Appl Radiat Isot 2008, 66: 340–345. 10.1016/j.apradiso.2007.09.007View ArticlePubMedGoogle Scholar
- Biechlin ML, D’Hardemare A, Fraysse M, Gilly FN, Bonmartin A: Improvement in radiolabelling proteins with the 99mTc-tricarbonyl-core [99mTc(CO)3]+, by thiol-derivatization with iminothiolane: application to c-globulins and annexin V. J Label Compd Radiopharm 2005, 48: 873–885. 10.1002/jlcr.999View ArticleGoogle Scholar
- Biechlin ML, Bonmartin A, Gilly FN, Fraysse M, D’Hardemare A: Radiolabeling of annexin A5 with 99mTc: comparison of HYNIC-Tc vs. iminothiolane-Tc-tricarbonyl conjugates. Nucl Med Biol 2008, 35: 679–687. 10.1016/j.nucmedbio.2008.05.009View ArticlePubMedGoogle Scholar
- Dias CR, Jeger S, Osso JA Jr, Müller C, De Pasquale C, Hohn A, Waibel R, Schibli R: Radiolabeling of rituximab with 188Re and 99mTc using the tricarbonyl technology. Nucl Med Biol 2011, 38: 19–28. 10.1016/j.nucmedbio.2010.05.010View ArticlePubMedGoogle Scholar
- Madsen J, Kristensen JB, Olsen OH, Christoffersen CL, Petersen LC, Tranholm M, Kjaer A, Hessea B: Recombinant coagulation factor VIIa labelled with the fac-99mTc(CO)3-core: synthesis and in vitro evaluation of a putative new radiopharmaceutical for imaging in acute bleeding lesion. J Label Compd Radiopharm 2010, 54: 214–219.View ArticleGoogle Scholar
- Amann A, Decristoforo C, Ott I, Wenger M, Bader D, Alberto R, Putz G: Surfactant protein B labelled with [99mTc(CO)3(H2O)3]+ retains biological activity in vitro . Nucl Med Biol 2001, 28: 243–250. 10.1016/S0969-8051(01)00192-5View ArticlePubMedGoogle Scholar
- Hofstrom C, Orlova A, Altai M, Wangsell F, Graslund T, Tolmachev V: Use of a HEHEHE purification Tag instead of a hexahistidine tag improves biodistribution of affibody molecules site-specifically labeled with 99mTc, 111In, and 125I. J Med Chem 2011, 54: 3817–3826. 10.1021/jm200065eView ArticlePubMedGoogle Scholar
- Tolmachev V, Hofström C, Malmberg J, Ahlgren S, Hosseinimehr SJ, Sandström M, Abrahmsén L, Orlova A, Gräslund T: HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [99mTc(CO)3]+, and shows improved biodistribution with reduced hepatic radioactivity accumulation. Bioconjug Chem 2010, 21: 2013–2022. 10.1021/bc1002357View ArticlePubMedGoogle Scholar
- Tavaré R, De Rosales R, Blower P, Mullen G: Efficient site-specific radiolabeling of a modified C2A domain of synaptotagmin I with [99mTc(CO)3]+: a new radiopharmaceutical for imaging cell death. Bioconjug Chem 2009, 20: 2071–2081. 10.1021/bc900160jView ArticlePubMedGoogle Scholar
- Tavaré R, Williams J, Howland K, Blower PJ, Mullen GE: [Re(CO)(3)](+) labelling of a novel cysteine/hexahistidine tag: insights into binding mode by liquid chromatography-mass spectrometry. J Inorg Biochem 2012, 114: 24–27.View ArticlePubMedGoogle Scholar
- Mees G, Dierckx R, Mertens K, Vermeire S, Van Steenkiste M, Reutelingsperger C, D’Asseler Y, Peremans K, Van Damme N, Van de Wiele C: 99mTc-labeled tricarbonyl his-CNA35 as an imaging agent for the detection of tumor vasculature. J Nucl Med 2012, 53: 464–471. 10.2967/jnumed.111.095794View ArticlePubMedGoogle Scholar
- Cortez-Retamozo V, Lahoutte T, Caveliers V, Olive L, Gainkam T, Hernot S, Packeu A, De Vos F, Vanhove C, Muyldermans S, De Baetselier P, Revets H: 99mTc-labeled nanobodies: a new type of targeted probes for imaging antigen expression. Curr Radiopharm 2008, 1: 37–41. 10.2174/1874471010801010037View ArticleGoogle Scholar
- Terán MA, Martínez E, Reyes AL, Paolino A, Vital M, Esperón P, Pacheco JP, Savio E: Biological studies in animal models using [99mTc](CO)3 recombinant annexin V as diagnostic agent of apoptotic processes. Nuc Med Biol 2010, 38: 279–285.View ArticleGoogle Scholar
- Orlova A, Nilsson FY, Wikman M, Widström C, Ståhl S, Carlsson J, Tolmachev V: Comparative in vivo evaluation of technetium and iodine labels on an anti-HER2 affibody for single-photon imaging of HER2 expression in tumors. J Nucl Med 2006, 47: 512–519.PubMedGoogle Scholar
- Deyev SM, Waibel R, Lebedenko EN, Schubiger AP, Plückthun A: Design of multivalent complexes using the barnase*barstar module. Nat Biotechnol 2003, 21: 1486–1492. 10.1038/nbt916View ArticlePubMedGoogle Scholar
- Zahnd C, Kawe M, Stumpp MT, de Pasquale C, Tamaskovic R, Nagy-Davidescu G, Dreier B, Schibli R, Binz HK, Waibel R, Plückthun A: Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size. Cancer Res 2010, 15: 1595–1605.View ArticleGoogle Scholar
- Berndorff D, Borkowski S, Moosmayer D, Viti F, Müller-Tiemann B, Sieger S, Friebe M, Hilger CS, Zardi L, Neri D, Dinkelborg LM: Imaging of tumor angiogenesis using 99mTc-labeled human recombinant anti-ED-B fibronectin antibody fragments. J Nucl Med 2006, 47: 1707–1716.PubMedGoogle Scholar
- Bidlingmaier S, He J, Wang Y, An F, Feng J, Barbone D, Gao D, Franc B, Broaddus VC, Liu B: Identification of MCAM/CD146 as the target antigen of a human monoclonal antibody that recognizes both epithelioid and sarcomatoid types of mesothelioma. Cancer Res 2009, 69: 1570–1577. 10.1158/0008-5472.CAN-08-1363PubMed CentralView ArticlePubMedGoogle Scholar
- Iyer AK, Lan X, Zhu X, Su Y, Feng J, Zhang X, Gao D, Seo Y, Vanbrocklin HF, Broaddus VC, Liu B, He J: Novel human single chain antibody fragments that are rapidly internalizing effectively target epithelioid and sarcomatoid mesotheliomas. Cancer Res 2011, 71: 2428–2432. 10.1158/0008-5472.CAN-10-3484PubMed CentralView ArticlePubMedGoogle Scholar
- Francis RJ, Mather JS, Chester K, Sharma SK, Bhatia J, Pedley RB, Waibel R, Green AJ, Begent RH: Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody- enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT). Eur J Nucl Med Mol Imaging 2004, 31: 1090–1096.PubMedGoogle Scholar
- De Saint-Hubert M, Mottaghy FM, Vunckx K, Nuyts J, Fonge H, Prinsen K, Stroobants S, Mortelmans L, Deckers N, Hofstra L, Reutelingsperger CP, Verbruggen A, Rattat D: Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo. Bioorg Med Chem 2010, 18: 1356–1363. 10.1016/j.bmc.2009.12.021View ArticlePubMedGoogle Scholar
- Willuda J, Kubetzko S, Waibel R, Schubiger PA, Zangemeister-Wittke U, Plückthun A: Tumor targeting of mono-, di-, and tetravalent anti-p185(HER-2) miniantibodies multimerized by self-associating peptides. J Biol Chem 2001, 276: 14385–14392.PubMedGoogle Scholar
- He J, Wang Y, Feng J, Zhu X, Lan X, Iyer AK, Zhang N, Seo Y, VanBrocklin HF, Liu B: Targeting prostate cancer cells in vivo using a rapidly internalizing novel human single-chain antibody fragment. J Nucl Med 2010, 51: 427–432. 10.2967/jnumed.109.069492PubMed CentralView ArticlePubMedGoogle Scholar
- Badar A, DeFreitas S, McDonnell JM, Yahya N, Thakor D, Razavi R, Smith R, Sacks S, Mullen GE: Recombinant complement receptor 2 radiolabeled with [99mTc(CO)3]+: a potential new radiopharmaceutical for imaging activated complement. PLoS One 2011, 6: e18275. 10.1371/journal.pone.0018275PubMed CentralView ArticlePubMedGoogle Scholar
- Emberson LM, Trivett AJ, Blower PJ, Nicholls PJ: Expression of an anti-CD33 single-chain antibody by Pichia pastoris. J Immunol Methods 2005, 305: 135–151. 10.1016/j.jim.2005.04.005View ArticlePubMedGoogle Scholar
- Maher J, Brentjens RJ, Gunset G, Riviere I, Sadelain M: Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta/CD28 receptor. Nat Biotechnol 2002, 20: 70–75. 10.1038/nbt0102-70View ArticlePubMedGoogle Scholar
- Strebe N, Guse A, Schüngel M, Schirrmann T, Hafner M, Jostock T, Hust M, Müller W, Dübel S: Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies. J Immunol Methods 2009, 341: 30–40. 10.1016/j.jim.2008.10.012View ArticlePubMedGoogle Scholar
- Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF: Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 1999, 112: 531–552.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.