
In-DOTAHis 6 -Z IGF1R:4551 . The use of 99m Tc instead of 111 In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His 6 In-DOTA-Z IGF1R:4551 . In NMRI nu/nu mice with DU-145 prostate cancer xenografts, the tumour uptake was 1.32±0.11 %ID/g and the tumour-to-blood ratio was 4.4±0.3 at 8 h after injection. The xenografts were visualized using a gamma camera 6 h after injection.
Conclusion
99m Tc(CO) 3 ]
Conclusion
The insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane tyrosine kinase involved in activation of mitogenic and antiapoptotic cellular signalling pathways [1] . Both preclinical and clinical data suggest that IGF-1R signalling acts as a prosurvival factor for several types of cancer, including breast, prostate, lung, colorectal, endometrial and bladder carcinomas and sarcomas, in which its upregulated expression confers resistance to different chemotherapeutic, hormonal and targeted therapies [1] [2] [3] [4] [5] . This is the rationale for targeting and deactivating IGF-1R as a potential approach to cancer therapy [6] . Several IGF-1R-targeting drugs are in preclinical and clinical development [7, 8] . The main strategies for deactivating signalling include receptor blockade, kinase inhibition and ligand sequestration. Currently, IGF-1R blockade by a monoclonal antibody is the most common approach in clinical trials [7] . Particularly in prostate cancer, inhibition of IGF-1R signalling is regarded as a promising methodology for the treatment of androgen-independent prostate cancer [9, 10] , because excessive signalling of IGF-1R leads to activation of androgen receptors in the absence of androgens, i.e. it causes androgen independence.
Conclusion
Reports from clinical trials emphasize that identification of predictive biomarkers and careful patient stratification are required for successful IGF-1R-targeted treatment [11] [12] [13] . Preclinical data demonstrate that a higher level of membranous IGF-1R is associated with response to IGF-1R inhibitors [14] . Clinical data also suggest that the response to the anti-IGF-1R antibody CP-751,871 correlates with high membrane expression of IGF-1R [15] . This creates a rationale for radionuclide molecular imaging of the extracellular domain of IGF-1R for patient stratification for therapy using blocking antibodies. Indeed, preclinical studies have demonstrated that imaging of IGF-1R can be used to predict the response to anti-IGF-1R antibody therapy of bone sarcoma [16] and IFG-1R-mediated resistance to trastuzumab therapy in breast cancer [17] .
Conclusion
It has to be noted that the level IGF-1R expression, which is associated with response to therapy, is rather low (10,000-30,000 receptors per cell) [18, 19] . In addition, there is normal physiological IGR-1R expression in a number of tissues, such as colon, lung, pancreas, salivary gland and stomach (http:// www.proteinatlas.org). A combination of low expression in tumours and ubiquitous expression in normal tissues makes radionuclide imaging of IGF-1R challenging and prompts the search for the optimal format of an imaging probe.
Conclusion
Affibody molecules are a relatively new class of targeting proteins based on protein A [20] . A small size (58 amino acids, 7 kDa) and usually high specificity and affinity (from low nanomolar to subnanomolar) make them attractive as imaging probes. Their excellent potential for radionuclide imaging of cancer-associated molecular targets has been shown in a number of preclinical studies [20] and a clinical study [21] . Tc would also allow a decreased dose burden to the patient, and to an appreciable reduction in the cost of imaging procedures. Furthermore, the previous generation of the imaging probe contained a hexahistidine (His 6 ) tag to facilitate purification of recombinantly produced Z IGF1R:4551 by immobilized metal ion affinity chromatography (IMAC). In a parallel study we have found that the use of the histidine-glutamatehistidine-glutamate-histidine-glutamate (HEHEHE) tag instead of a His 6 tag permits purification of anti-HER2 Affibody molecules by IMAC, enables their convenient site-specific labelling with [ , and improves their biodistribution by reducing hepatic accumulation of radioactivity [23] . We hypothesized that replacement of the His 6 tag with a HEHEHE tag in Z IGF1R:4551 would similarly permit convenient purification using IMAC, enable labelling with [ 
Materials and methods

Materials 99m
Tc was obtained as pertechnetate from an UltraTechneKow generator (Covidien) by elution with 0.9 % NaCl. The IsoLink kit was kindly provided by Covidien. Radioactivity was measured using a gamma-counter with a 3-inch NaI(Tl) detector (1480 WIZARD; PerkinElmer). The purity of nonlabelled Affibody molecules was determined using reverse-phase high-performance liquid chromatography (RP-HPLC). The purity of radiolabelled Affibody molecules was determined by radio-ITLC (150-771 Tec-Control Chromatography; Biodex Medical Systems) cross-validated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The distribution of radioactivity along ITLC strips and SDS-PAGE gels was measured on a Cyclone™ Storage Phosphor System (PerkinElmer). The purity of all studied compounds was over 95 %. Data on cellular uptake and biodistribution were evaluated by an unpaired, two-tailed t-test using the GraphPad Prism program (GraphPad Software, La Jolla, CA) to determine the significance of differences (p<0.05).
Materials 99m
Production, purification and characterization of the (HE) 3 -Z IGF1R:4551 Affibody molecule The (HE) 3 -Z IGF1R:4551 Affibody molecule was produced and purified essentially as previously described [24] . Briefly, the PCR fragment encoding for (HE) 3 -Z IGF1R:4551 was subcloned into the expression vector pET21a(+) (Novagen, Germany) and the correct DNA sequence was verified by DNA sequencing. The Affibody molecule was expressed in Escherichia coli strain BL21(DE3) and was recovered by IMAC using a Talon metal affinity resin (BD Bioscience, San Jose, CA) as previously described [25] .
Materials 99m
The (HE) 3 -Z IGF1R:4551 Affibody molecule was further purified by RP-HPLC on an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA). The sample was injected onto a C18 column at 20 % B-buffer (the rest was A-buffer) and eluted using a 20 min gradient from 20-65 % B-buffer (A: 0.1 % trifluoroacetic acid/H 2 O, B: 0.1 % trifluoroacetic acid/ CH 3 CN), at a flow rate of 2.5 ml/min. The final purity of (HE) 3 -Z IGF1R:4551 was determined by analytical RP-HPLC as previously described [24] . The purity was determined by dividing the area of the peak corresponding to the Affibody molecule with the total area of all peaks in the chromatogram. The protein concentration was determined by amino acid analysis (Amino Acid Analysis Center, Uppsala University).
Circular dichroism
Circular dichroism spectra of (HE) 3 -Z IGF1R:4551 at a concentration of 60 μM in PBS were recorded from 250 to 195 nm at 20°C using a spectropolarimeter (J-810; JASCO, Tokyo, Japan). For measurement over a range of temperatures, the absorbance at 221 nm was recorded as a function of increasing temperature from 20°C to 90°C (5°C/min).
Biosensor analysis
Affinity constants were determined by surface plasmon resonance (SPR) analysis on a Biacore 2000 instrument (GE Healthcare, Uppsala, Sweden) using a single-cycle kinetic method. The extracellular domains of human and mouse IGF-1R (R&D Systems, Minneapolis, MN) were immobilized (600 RU and 1500 RU, respectively) on different flow-cell surfaces of a CM5 chip (Biacore Life Science, GE Healthcare) using amine coupling, according to the manufacturer's instructions. A fourfold dilution series comprising five concentrations was prepared and consecutively injected over the flow-cell surfaces, with increasing concentrations ranging from 1.31 to 336 nM at a flow rate of 30 μl/min. The sensorgrams obtained were initially analysed using Biacore evaluation software (Biacore Life Science, GE Healthcare). For detailed analysis, the sensorgrams were subjected to distribution-based interaction analysis using the Interaction Map tool [26] in TraceDrawer 1.4 (Ridgeview Instruments, Uppsala, Sweden).
Biosensor analysis
Labelling and in vitro stability (HE) 3 -Z IGF1R:4551 was labelled with [ 99m Tc(CO) 3 ] + as described previously [27] . Briefly, 500 μl (200-320 MBq) of 99m TcO 4 − -containing generator eluate was added to a vial with the IsoLink kit. The mixture was incubated at 100°C for 20 min. Thereafter, 40 μl of the mixture was transferred to a tube containing 50 μg (about 6.8 nmol) Affibody molecule in 40 μl PBS and incubated at 50°C for 60 min. The labelled Affibody molecule was purified using size exclusion NAP-5 columns (GE Healthcare), preequilibrated and eluted with PBS. The purity of each preparation was evaluated using radio-ITLC.
Biosensor analysis
To predict in vivo stability, two samples of [ For blocking, a 100-fold excess of nonlabelled (HE) 3 -Z IGF1R:4551 was added 15 min before the radiolabelled conjugate to saturate the receptors. The cells were incubated for 2 h in a humidified incubator at 37°C. The medium was then collected, the cells were detached with a trypsin-EDTA solution (0.25 % trypsin, 0.02 % EDTA; Biochrom, Berlin, Germany), and the radioactivity in the cells and medium was measured to determine the cell-bound radioactivity fraction.
Biosensor analysis
The rate of internalization of [ 99m Tc(CO) 3 ] + -(HE) 3 -Z IGF1R:4551 by DU-145 cells was measured according to a previously described method [22] . Briefly, the cells (approximately 10 6 per dish) were incubated with the labelled compound (1.5 nM) at 37°C. At predetermined time points (1, 2, 3, 4, 8 and 24 h after the start of incubation), the medium from a set of three dishes was removed. To collect the membranebound radioactivity, the cells were treated with 0.2 M glycine buffer containing 4 M urea, pH 2.5, for 5 min on ice. To collect the radioactivity internalized by the cells, the cells were treated with 1 M NaOH at 37°C for 0.5 h. The percentage of internalized radioactivity was calculated for each time point.
Biosensor analysis
To measure the affinity of [ 99m Tc(CO) 3 ] + -(HE) 3 -Z IGF1R:4551 for DU-145 cells, the cells were incubated for 4 h at 4°C with radiolabelled Affibody molecule at concentrations in the range 0.08 to 27 nM in complete medium. For each data point, four dishes were used, including one presaturated with unlabelled (HE) 3 -Z IGF1R:pauladavid@example.net μM to determine nonspecific binding. After incubation, the medium was aspirated, and the cells were detached with trypsin-EDTA solution and counted using an automatic cell counter (Beckman Coulter). The radioactivity of the samples was measured. The data were analysed using GraphPad Prism software (GraphPad Software, San Diego, CA).
In vivo studies
All animal experiments were planned and performed in accordance with national legislation on protection of laboratory animals and were approved by the Ethics Committee for Animal Research in Uppsala. For xenografting, 5×10 6 DU-145 cells (in Matrigel®; BD Biosciences; 50 % in PBS) were subcutaneously implanted in the right hind leg of Balb/C nu/ nu mice. Xenografts were allowed to grow for 2 weeks. 111 In-DOTA-His 6 -Z IGF1R:4551 (10 kBq per mouse) in 100 μl PBS was intravenously injected into male NMRI mice (average weight 33.5± 2.4 g). The total injected protein dose was adjusted by dilution with nonlabelled (HE) 3 -Z IGF1R:4551 to 1 μg per mouse. At 1, 4 and 24 h p.i., a group of four mice were killed by injection of a lethal dose of anaesthetic (20 μl of solution per gram of body weight; 50 mg/ml, Ketalar, Pfizer; 20 mg/ml, Rompun, Bayer) followed by heart puncture and exsanguination with a syringe rinsed with heparin (5,000 IE/ml, Leo Pharma). Samples of blood, colon, lung, liver, spleen, pancreas, kidney, muscle and bone were collected and weighed, and their radioactivity was measured. In addition, the gastrointestinal tract with its contents was collected to estimate hepatobiliary excretion of radioactivity. The whole gamma spectrum was recorded for each sample. In in the range 150-480 keV. The data were corrected for dead time, background and spillover. Tissue uptake was calculated as percent of the injected dose per gram (% ID/g). A paired t-test was used to evaluate the significance of differences (p<0.05) in biodistribution of the conjugates. , 1 μg, in 100 μl PBS) and imaging was performed 6 h later. Immediately before imaging, the animals were killed by an overdose of anaesthetic. After the animals had been killed, the urinary bladders were excised. Imaging was performed using a GE Infinia gamma camera equipped with a low-energy high-resolution collimator. Static images (30 min) were obtained with a zoom factor of 2 in a 256×256 matrix. The images were evaluated using Osiris 4.19 software (University Hospital of Geneva, Switzerland). In each animal, a region of interest was drawn around the tumour. The same region was copied to the contralateral thigh. Tumour-to-contralateral thigh ratios were calculated based on average counts in the regions of interest.
Results
Production, purification and characterization of the (HE) 3 -Z IGF1R:4551 Affibody molecule (HE) 3 -Z IGF1R:4551 was recombinantly expressed in E. coli under control of the T7 promoter. After harvest, the lysate containing (HE) 3 -Z IGF1R:4551 was heat-treated for 10 min at 60°C to precipitate a proportion of endogenous E. coli proteins prior to IMAC purification on an affinity resin containing immobilized Co 2+ . The (HE) 3 -Z IGF1R:4551 Affibody molecule was successfully recovered by IMAC, and was further purified by RP-HPLC. Analysis by SDS-PAGE of a sample taken after RP-HPLC showed only a single band with a molecular weight corresponding to the expected molecular mass of (HE) 3 -Z IGF1R:4551 (Fig. 1a) . LC-MS analysis confirmed the molecular weight of the recovered protein (calculated molecular weight of (HE) 3 -Z IGF1R:4551 is 7,463 Da; found molecular weight was 7,464 Da; Fig. 1c) , and RP-HPLC analysis demonstrated exceptional purity (99 %; Fig. 1b) .
Results
The secondary structure of (HE) 3 -Z IGF1R:4551 was investigated by circular dichroism. As expected, (HE) 3 -Z IGF1R:4551 gave a circular dichroism spectrum showing a high alphahelical content (Fig. 2a) , similar to the spectrum recorded previously for the similar Affibody construct (His 6 )-Z IGF1R:4551 [22] . The spectra recorded for (HE) 3 -Z IGF1R:4551 before and after heating to 90°C were also highly similar, indicating that the construct was able to completely refold after thermal denaturation. The melting temperature of (HE) 3 -Z IGF1R:4551 was determined as 58°C (Fig. 2b) .
Results
The interaction kinetics between (HE) 3 -Z IGF1R:4551 and human and mouse IGF-1R were analysed on a Biacore 2000 instrument using single-cycle injection. Evaluation of the sensorgrams did not reflect a one-to-one interaction event between (HE) 3 -Z IGF1R:4551 and the receptors. A heterogeneous ligand interaction model, indicating the occurrence of two independent binding events, reflected the observed interaction better, but not sufficiently well. Interaction Map analysis revealed two well-defined processes for both receptors. For the human receptor (Fig. 2c) was efficient, with a yield of 69±7 %. Simple purification using a disposable size-exclusion column resulted in a radiochemical purity of 99.1±0.9 %. An isolated radiochemical yield of 64±6 % was achieved. Figure 3 shows the result of a stability test in which [ 99m Tc(CO) 3 ] + -(HE) 3 -Z IGF1R:4551 was found to be very stable with a purity of 97.7±0.6 % after a 1-h incubation in murine serum at 37°C. + -(HE) 3 -Z IGF1R:4551 to the cells was 1.7 nM (Fig. 5) . Fig. 6 . Biodistribution of both compounds was characterized by rapid renal clearance from blood and normal tissues. Radioactivity accumulation in the gastrointestinal tract (a measure for hepatobiliary excretion) was less than 7 % of the injected radioactivity for both conjugates (data not shown). Blood clearance of 111 In was slightly faster than that of 99m Tc. Clearance from the body was accompanied by renal reabsorption of radioactivity. The kidney uptake of 99m Tc was 1.3-fold higher at 1 h p.i., but at later time points (4 and 24 h p.i.), the renal retention of 111 In was higher. Generally, the clearance rate of 99m Tc was higher than that of 111 In for the majority of organs; however, the difference was most pronounced at 24 h p.i. The greatest difference in radioactivity uptake was seen in the liver and spleen. 111 In uptake in these organs was 3.6-fold higher than that of 99m Tc at 4 h p.i., and the difference was greater at 24 h p.i. Fig. 7 . The tracer demonstrated rapid first phase clearance from blood. Clearance of radioactivity from the pancreas, stomach, colon and muscles was more rapid than clearance from tumour. Tumour-to-blood, tumour-to-muscle and tumour-tocolon ratios had a tendency to increase from 4 to 8 h p.i. (Fig. 8 ), but the difference was small. The optimal imaging time using (Fig. 9) , confirming targeting specificity. Preinjection of a large excess of (HE) 3 -Z IGF1R:4551 significantly (p<0.05) Fig. 11 . The tumour-to-contralateral side ratio was 3.9±0.1. The kidneys, liver and salivary glands were clearly visualized, indicating substantial accumulation of radioactivity as predicted from the biodistribution studies described above.
In vivo studies

Discussion
Clinical trials using IGF-1R-blocking monoclonal antibodies have shown that there is an urgent unmet clinical need for the development of predictive biomarkers permitting patient selection for such therapy [28] . Pretreatment concentrations of IGF-1 in serum [12, 13] , expression of IGF-1R in tumours, and levels of circulating IGF-2 and IGF-binding proteins [12] have all been evaluated as predictors of response to IGF-1R-targeting therapy in clinical trials. The level of membrane IGF-1R expression have been found to be correlated to the response rate to therapy using the antibody CP-751,871 [15] . Standard procedures for determining biomarkers, including blood sampling and biopsy-based methods, cannot appropriately address heterogeneity of target expression. In addition, repeated invasive biopsy acquisition is preferably avoided. Preclinical data also suggest that the level of IGF-1R expression may change over time [29] , as targeting therapy may cause a loss of receptor expression in cancer cells and induce tumour proliferation due to activation of other pathways. Such a switch is difficult to detect by biopsy-based methods. In contrast, radionuclide imaging may provide information on IGF-1R expression at different sites (including all metastases) in a single scan, from which conclusions about expression heterogeneity may be drawn. This noninvasive procedure can be performed repeatedly, allowing changes in expression level in response to therapy to be followed.
Discussion
The use of a therapeutic antibody as a targeting agent is an obvious choice for radionuclide imaging. In this way, not only the level of the molecular target in the tumour might be evaluated, but also its accessibility to the proposed therapy [16, 30, 31] . However, slow clearance from blood and organs in equilibrium with blood requires several (3 to 7) days between injection of the probe and measurement to obtain a reasonably high tumour-to-blood ratio. In contrast, the use of smaller targeting agents such as Affibody molecules achieves the same tumour-to-blood ratio on the day of injection [22] . However, the rapid clearance of small targeting proteins reduces their bioavailability, causing low absolute uptake in the tumour. To obtain a higher signal from the tumour, a higher injected activity is desirable. However, the dosimetric properties of 111 In, which was used for labelling of the first generation of anti-IGF-1R Affibody molecules [22] , put a limitation on the amount of injected radioactivity. The switch to 99m Tc in this study makes it possible to inject a higher dose of radioactivity due to the appreciably shorter half-life and lower locally absorbed dose. Furthermore, the properties of the gamma radiation emitted by 99m Tc permit the use of lowenergy collimators, which increases resolution and sensitivity of imaging. Thus, 99m Tc is a better label for IGF-1R-targeting Affibody molecules than 111 In.
Discussion
Previous studies using a HER2-targeting Affibody molecules suggest that the two most promising approaches to sitespecific 99m Tc labelling are to couple [ 99m Tc(CO) 3 ] + to a histidine-containing motif, for example the HEHEHE tag [23] , and to couple (Tc0O) 3+ to a cysteine-containing peptide-based chelator containing, for example, the motif GGGC at the C terminus [32] . In both cases the chelator is built into the sequence of the targeting protein, which allows production in a single biotechnological process avoiding the need for further conjugation steps. For a construct containing the HEHEHE tag, a convenient IMAC purification may be combined with low liver accumulation of radioactivity, and the use of a GGGC motif as chelator causes low radioactivity accumulation in the kidneys, but requires more complicated purification schemes during tracer production [33] . A combination of these approaches is tempting, but recent data suggest that coupling of (Tc0O) 3+ to a GGGC motif in the presence of a HEHEHE tag in the probe is associated with loss of site specificity of labelling and poor control over biodistribution [34] . In this study, we therefore evaluated a labelling approach in which [ 99m Tc(CO) 3 ] + was conjugated to the HEHEHE tag. Affibody molecules are a relatively new kind of targeting protein. Although different factors influencing the efficacy of radionuclide imaging are relatively well studied [20, 35, 36] , the majority of the parameters were obtained using derivatives of a single Affibody variant, the anti-HER2 Z HER2:342 Affibody molecule. It is not obvious that this information can be directly translated to the development of other Affibody molecules. However, the results of this study show that similar results may also be obtained with an Affibody molecule binding to a different target, the IGF-1R. The introduction of a HEHEHE tag permitted efficient production and purification of Z IGF1R:4551 while the high affinity to IGF-1R was retained according to SPR evaluation results (Fig. 2c) .
Discussion
The SPR evaluation of Z IGF1R:4551 binding to IGF-1R showed two parallel processes with different kinetics for human and mouse receptors (Fig. 2c, d ). The cell-based binding in vitro assay also showed that the kinetics of binding to the human receptor were biphasic (Fig. 4b) . This suggests that Z IGF1R:4551 binds to two different binding sites on IGF-1R. This hypothesis is supported by the fact IGF-1R is a dimer with two binding sites, and the binding of the natural ligand, IGF-1, shows negative cooperativity [35] . The parental Affibody molecule to Z IGF1R:4551 was selected by phage display to compete with the receptor's natural ligand IGF-1 [37] , and negative cooperativity could also be expected in this case.
Discussion
The HEHEHE tag allowed efficient and stable labelling of (HE) 3 + using a commercial IsoLink kit (Fig. 3) . [ (Fig. 4) and low nanomolar affinity in binding to living IGF-1R-expressing cells (Fig. 5 ). An interesting feature of [ Affibody molecules was its more rapid internalization by cancer cells (Fig. 4) . This suggests that the use of residualizing labels is desirable for efficient accumulation of radioactivity in the tumours.
Discussion
A Tclabelled variant (Fig. 6) . In particular, uptake of (Fig. 9) . This experiment also confirmed IGF-1R-specific accumulation of radiolabelled Z IGF1R:4551 in IGF-1R-expressing organs, including the lung, pancreas, stomach and salivary gland (Fig. 9 ). This suggests that the murine model is adequate for in vivo studies of targeting of IGF-1R using (HE) 3 -Z IGF1R:4551 because it takes into account specific interactions with host tissues. The suitability of the model is further supported by the SPR analyses (Fig. 2c, d) , which showed the affinity of (HE) 3 In-DOTAHis 6 -Z IGF1R:4551 was performed at 6 h p.i (Fig. 9) . [ In also significantly increased the tumour-to-lung, tumourto-liver, tumour-to-spleen, tumour-to-pancreas and tumour-tosalivary gland ratios (Fig. 10) .
Discussion
In conclusion, the [ 99m Tc(CO) 3 ] + -(HE) 3 -Z IGF1R:4551 Affibody molecule is a promising agent for visualization of IGF-1R in malignant tumours. Its use might enable patient stratification for IGF-1R-targeting therapy allowing more individualized cancer treatment.

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