Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount.

INTRODUCTION: Overexpression of epidermal growth factor receptor (EGFR) is a prognostic and predictive biomarker in a number of malignant tumours. Radionuclide molecular imaging of EGFR expression in cancer could influence patient management. However, EGFR expression in normal tissues might complicate in vivo imaging. The aim of this study was to evaluate if optimization of the injected protein dose might improve imaging of EGFR expression in tumours using a novel EGFR-targeting protein, the DOTA-Z(EGFR:2377) Affibody molecule. METHODS: An anti-EGFR Affibody molecule, Z(EGFR:2377), was labelled with (111)In via the DOTA chelator site-specifically conjugated to a C-terminal cysteine. The affinity of DOTA-Z(EGFR:2377) for murine and human EGFR was measured by surface plasmon resonance. The cellular processing of (111)In-DOTA-Z(EGFR:2377) was evaluated in vitro. The biodistribution of radiolabelled Affibody molecules injected in a broad range of injected Affibody protein doses was evaluated in mice bearing EGFR-expressing A431 xenografts. RESULTS: Site-specific coupling of DOTA provided a uniform conjugate possessing equal affinity for human and murine EGFR. The internalization of (111)In-DOTA-Z(EGFR:2377) by A431 cells was slow. In vivo, the conjugate accumulated specifically in xenografts and in EGFR-expressing tissues. The curve representing the dependence of tumour uptake on the injected Affibody protein dose was bell-shaped. The highest specific radioactivity (lowest injected protein dose) provided a suboptimal tumour-to-blood ratio. The results of the biodistribution study were confirmed by gamma-camera imaging. CONCLUSION: The (111)In-DOTA-Z(EGFR:2377) Affibody molecule is a promising tracer for radionuclide molecular imaging of EGFR expression in malignant tumours. Careful optimization of protein dose is required for high-contrast imaging of EGFR expression in vivo.

Affibody molecule-mediated depletion of HSA and IgG using different buffer compositions: a 15 min protocol for parallel processing of 1-48 samples.

High-abundant plasma proteins pose a challenge in a large number of proteomics-based technologies. Depletion of these high-abundant proteins has proven to be a fruitful strategy to circumvent masking of lower-abundant proteins that could serve as valuable biomarker candidates. However, current strategies often do not meet the throughput requirements of large-scale proteomic studies. In the present paper, a flexible and parallelized method for the depletion of high-abundant proteins is described, allowing the removal of the two most abundant proteins from 48 blood-derived samples in less than 15 min using Affibody molecules as affinity ligands. A sample-processing platform like this should be suitable for a number of proteomics technologies; its flexibility in buffer composition allows for different types of downstream applications.

Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging.

PURPOSE: The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling both recombinant and synthetic production and site-specific labelling with (99m)Tc or trivalent radiometals. METHODS: The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow (99m)Tc labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium labelling. The resulting (99m)Tc- and (111)In-labelled Affibody molecules were evaluated both in vitro and in vivo. RESULTS: Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour to blood ratio in LS174T xenografts was 30 at 4 h post-injection for both conjugates. Biodistribution data showed that the (99m)Tc-labelled Affibody molecule had a fourfold lower kidney accumulation compared with the (111)In-labelled Affibody molecule while the accumulation in other organs was similar. Gamma camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. CONCLUSION: Incorporation of the C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt or yttrium) and for a flexible production (chemical synthesis or recombinant).

Evaluation of a maleimido derivative of CHX-A'' DTPA for site-specific labeling of affibody molecules.

Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule Z HER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A'' DTPA was site-specifically conjugated at the C-terminal cysteine of Z HER2:2395-C, a variant of Z HER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with (111)In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of (111)In-CHX-A'' DTPA-Z 2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of (111)In-CHX-A'' DTPA-Z 2395-C was 17.3 +/- 4.8% IA/g and the tumor-to-blood ratio 86 +/- 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A'' DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.

Production of milligram quantities of affinity tagged-proteins using automated multistep chromatographic purification.

A new chromatography system, AKTAxpress (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) has been designed to meet the demand for high-throughput purification of proteins in structural genomics and drug discovery. The system offers a number of automated multistep purification protocols for affinity-tagged proteins. All protocols start with affinity chromatography followed by combinations of desalting, ion exchange chromatography and gel filtration. As an option, tag removal can be included in the purification protocols. Up to 16 proteins can be purified per day and the yield can be as high as 50 mg of each protein at > 90% purity. To highlight the versatility of the system, this paper presents several case studies; purifications of hexahistidine- and glutathione S-transferase-tagged proteins using different protocols, automated on-column tag cleavage and optimization studies for a hexahistidine-tagged kinase.

Improving the tolerance of a protein a analogue to repeated alkaline exposures using a bypass mutagenesis approach.

Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.

Similar Listeria monocytogenes pulsotypes detected in several foods originating from different sources.

The purpose of the study was to obtain fingerprinting data of Listeria monocytogenes strains isolated in various foods to determine possible associations of strains with product type, producer, country or isolation time. Two hundred and ninety-five L. monocytogenes strains originating from food items of 41 producers of 10 countries were characterized by pulsed-field gel electrophoresis (PFGE) typing. Combination of AscI and ApaI macrorestriction patterns (MRP) yielded 66 different pulsotypes. Ten pulsotypes were common to two or more product types and 17 pulsotypes were detected in foods of more than one producer having no apparent association with each other. Similar pulsotypes of L. monocytogenes were recovered in products of different countries over several years. Some of the pulsotypes were recurrently recovered from the same product of the same producer, suggesting a possible persistence of these strains in the processing plant. However, some of the recurrently isolated L. monocytogenes pulsotypes were repeatedly found in products of several producers, which may indicate that persistent houseflora strains are not always producer-specific. Furthermore, the similarity of macrorestriction patterns expressed as clusters, based on the numerical analysis of macrorestriction patterns, was not found to correlate with product type, country, producer or year of isolation. Our data suggest a wide geographical and temporal distribution of a number of L. monocytogenes strains isolated in food products. The existence of similar L. monocytogenes strains in various food products of several producers should be considered if food strain fingerprint results are used to help trace the vehicles for infections.

Characterization of Listeria monocytogenes isolates from the meat, poultry and seafood industries by automated ribotyping.

A total of 564 Listeria monocytogenes isolates were characterized by automated ribotyping. The samples were taken from equipment, personnel and the environment after cleaning procedures and during food processing, as well as from raw materials and products from six meat, two poultry and five seafood processing plants located in the Faroe Islands, Finland, Iceland, Norway and Sweden. Altogether, 25 different ribotypes (RTs) were generated. Two RTs occurred in the samples from all three food sectors--meat, poultry and seafood. Four RTs occurred in meat and poultry plant samples and other four RTs occurred in meat and seafood plant samples. Five RTs occurred only in meat plant samples, five only in poultry plant samples and five only in seafood plant samples. Eight of the thirteen plants had their own in-house L. monocytogenes ribotype. There was geographical differences between the RTs, but no correlation between RTs and food sectors was detected. The discrimination power of automated ribotyping was satisfactory to trace the contamination sources in the food processing plants clearly indicating the sites at which improved cleaning procedures were necessary. In addition, it was possible to screen a large number of isolates with two instruments located at different institutes and to make a reliable combination of the results.

Typing of Listeria monocytogenes isolates originating from the food processing industry with automated ribotyping and pulsed-field gel electrophoresis.

A total of 486 Listeria monocytogenes isolates originating from 17 Finnish food processing plants (representing meat, poultry, fish, and dairy production) were collected and typed by automated ribotyping using EcoRI as the restriction enzyme. The isolates were divided into 16 different ribotypes (RTs). Some of these isolates (121), representing all EcoRI types and 16 food plants, were subjected to ribotyping with the PvuII enzyme, to pulsed-field gel electrophoresis (PFGE) typing with AscI and SmaI restriction enzymes, and to serotyping with O-antigen antisera. Nineteen ribotypes were generated with PvuII, 42 macrorestriction patterns were generated with AscI and 24 with SmaI, and three serotypes were generated with antisera. When the results were combined, the overall number of RTs was 23, and that of the PFGE types was 46. Thus, the overall discrimination power of PFGE was higher (discrimination index [DI] 0.966) than that of ribotyping (DI 0.906). The most common serotype (90.1% of the isolates) was 1/2, and isolates of serotype 4 (3.3%) were rare. There was no connection between food sectors and RTs or PFGE types, but PFGE indicated the single plants (78.3% of the types) better than ribotyping (56.5%). On the basis of its automation and on the availability of identification databases, automated ribotyping had some advantages over PFGE. Overall, automated ribotyping can be considered a practical and rapid tool when Listeria contamination is suspected and when screening a large number of isolates is necessary, e.g., when tracing contamination sources. However, in cases of outbreaks, the identical patterns must be confirmed by PFGE, which is a more discriminatory method.

Validation of the Hygicult E dipslides method in surface hygiene control: a Nordic collaborative study.

A collaborative study with Enterobacteriaceae was conducted to validate Hygicult E dipslides by comparison with violet red bile glucose agar (VRBGA) contact plates and swabbing, using stainless steel surfaces artificially contaminated with microbes at various levels. Twelve laboratories participated in the validation procedure. The total number of collaborative samples was 108. The microbial level in each sample was assessed in triplicate by using the 3 above-mentioned methods. No Enterobacteriaceae were used at the low inoculation level. At the middle inoculation level, the percentages detached from the test surfaces were 16.6 with the Hygicult E method, 15.3 with the contact plate method, and 14.6 with swabbing; at the high innoculation level, the percentages were 14.5, 15.8, and 9.8, respectively. The percentage of acceptable results after the removal of outliers was 97.2. Repeatability relative standard deviations ranged from 33.4 to 44.9%; reproducibility relative standard deviations ranged from 45.2 to 77.1%. The Hygicult E dipslide, VRBGA contact plate, and swabbing methods gave similar results at all 3 microbial levels tested: <1.0 colony-forming units (CFU)/cm2 at the low level, 1.2-1.3 CFU/cm2 at the middle level (theoretical yield 8.0 CFU/cm2), and 1.2-2.0 CFU/cm2 at the high level (theoretical yield 12.5 CFU/cm2).

Tumor targeting using affibody molecules: interplay of affinity, target expression level, and binding site composition.

UNLABELLED: Radionuclide imaging of cancer-associated molecular alterations may contribute to patient stratification for targeting therapy. Scaffold high-affinity proteins, such as Affibody molecules, are a new, promising class of probes for in vivo imaging. METHODS: The effects of human epidermal growth factor receptor 2 (HER2) affinity and binding site composition of HER2-binding Affibody molecules, and of the HER2 density on the tumor targeting, were studied in vivo. The tumor uptake and tumor-to-organ ratios of Affibody molecules with moderate (dissociation constant [K(D)] = 10(-9) M) or high (K(D) = 10(-10) M) affinity were compared between tumor xenografts with a high (SKOV-3) and low (LS174T) HER2 expression level in BALB/C nu/nu mice. Two Affibody molecules with similar affinity (K(D) = 10(-10) M) but having alternative amino acids in the binding site were compared. RESULTS: In SKOV-3 xenografts, uptake was independent of affinity at 4 h after injection, but high-affinity binders provided 2-fold-higher tumor radioactivity retention at 24 h. In LS174T xenografts, uptake of high-affinity probes was already severalfold higher at 4 h after injection, and the difference was increased at 24 h. The clearance rate and tumor-to-organ ratios were influenced by the amino acid composition of the binding surface of the tracer protein. CONCLUSION: The optimal affinity of HER2-binding Affibody molecules depends on the expression of a molecular target. At a high expression level (>10(6) receptors per cell), an affinity in the low-nanomolar range is sufficient. At moderate expression, subnanomolar affinity is desirable. The binding site composition can influence the imaging contrast. This information may be useful for development of imaging agents based on scaffold affinity proteins.

Imaging of insulinlike growth factor type 1 receptor in prostate cancer xenografts using the affibody molecule 111In-DOTA-ZIGF1R:4551.

UNLABELLED: One of the pathways leading to androgen independence in prostate cancer involves upregulation of insulinlike growth factor type 1 receptor (IGF-1R). Radionuclide imaging of IGF-1R in tumors might be used for selection of patients who would most likely benefit from IGF-1R-targeted therapy. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of IGF-1R expression in prostate cancer xenografts using a small nonimmunoglobulin-derived binding protein called an Affibody molecule. METHODS: The IGF-1R-binding Z(IGF1R:4551) Affibody molecule was site-specifically conjugated with a maleimido derivative of DOTA and labeled with (111)In. The binding of radiolabeled Z(IGF1R:4551) to IGF-1R-expressing cells was evaluated in vitro and in vivo. RESULTS: DOTA-Z(IGF1R:4551) can be stably labeled with (111)In with preserved specific binding to IGF-1R-expressing cells in vitro. In mice, (111)In-DOTA-Z(IGF1R:4551) accumulated in IGF-1R-expressing organs (pancreas, stomach, lung, and salivary gland). Receptor saturation experiments demonstrated that targeting of DU-145 prostate cancer xenografts in NMRI nu/nu mice was IGF-1R-specific. The tumor uptake was 1.1 ± 0.3 percentage injected dose per gram, and the tumor-to-blood ratio was 3.2 ± 0.2 at 8 h after injection. CONCLUSION: This study demonstrates the feasibility of in vivo targeting of IGF-1R-expressing prostate cancer xenografts using an Affibody molecule. Further development of radiolabeled Affibody molecules might provide a useful clinical tool for stratification of patients with prostate cancer for IGF-1R-targeting therapy.

Application of radiochemical determination methods in cleanability research of building materials.

During recent years increasing effort has been made to modify surface properties with easy-to-clean or self-cleaning characteristics, and concomitantly there is a need to be able to quantify cleanability. Methodology is a complex issue, including aspects of selection and characterization of the surface materials, the soiling materials (contaminants), soiling and cleaning methods, and the detection methods. Different biological, chemical, physical and visual methods have been included in studies of surface cleanability. One challenge has been to obtain quantitative information about soiling. The radiochemical methods, gamma spectrometry (NaI(Tl)-crystal) and liquid scintillation counting, have been shown to be suitable for evaluating cleanability of different surface materials and different soiling material types, providing quantitative information about the amount of soiling material both on and beneath the surface. Due to the different labelled soiling components, the interaction of the surface with different soiling material types can be evaluated. Radiochemical methods have unique benefits particularly for examining porous materials and surfaces. However, they are suitable only for highly controlled studies because of the hazards. Different features and details of radiochemical methods are discussed with the view to aid planning of future cleanability studies.

Generation of monospecific antibodies based on affinity capture of polyclonal antibodies.

A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

Design of an optimized scaffold for affibody molecules.

Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K(D), of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.

Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules.

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.

Affibody-mediated transferrin depletion for proteomics applications.

An Affibody (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.