Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.

BACKGROUND: Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. METHODS: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377-ST and ZTaq:3638-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with (11)C for in vivo PET studies. Kinetic scans with the (11)C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. RESULTS: Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZEGFR:2377-ST-DyLight488. [Methyl-(11)C]-labeled ZEGFR:2377-ST-CH3 and ZTaq:3638-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-(11)C]-ZEGFR:2377-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since ZEGFR:2377 binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. CONCLUSIONS: Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and (11)C-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.

188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment.

UNLABELLED: Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of (99m)Tc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate (188)Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors. METHODS: ZHER2:V2 was labeled with (188)Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment. RESULTS: Binding of (188)Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that (188)Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible. CONCLUSION: (188)Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.

Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

Design and evaluation of radiolabeled tracers for tumor imaging.

The growing understanding of tumor biology and the identification of tumor-specific genetic and molecular alterations, such as the overexpression of membrane receptors and other proteins, allows for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools used to identify patients who are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising noninvasive method to visualize and characterize the expression of such targets. A number of different proteins, from full-length antibodies and their derivatives to small scaffold proteins and peptide receptor-ligands, have been applied to molecular imaging, each demonstrating strengths and weaknesses. Here, we discuss the concept of molecular targeting and, in particular, molecular imaging of cancer-associated targets. Additionally, we describe important biotechnological considerations and desired features when designing and developing tracers for radionuclide molecular imaging.

Generation and evaluation of bispecific affibody molecules for simultaneous targeting of EGFR and HER2.

Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly(4)Ser)(3) or (Ser(4)Gly)(3), and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser(4)Gly)(3) linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly(4)Ser)(3). It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.

HER2-positive tumors imaged within 1 hour using a site-specifically 11C-labeled Sel-tagged affibody molecule.

UNLABELLED: A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. METHODS: A HER2-binding Affibody molecule, Z(HER2:342), was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either (11)C or (68)Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the (11)C-labeled Affibody molecule. RESULTS: Both the (11)C- and (68)Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4-5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (>5-fold) for the (11)C-labeled protein, and its overall absorbed dose was considerably lower. (11)C-Z(HER2:342) showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P = 0.002), comparable to uptake in tumors with low HER2 expression. CONCLUSION: To our knowledge, the Sel-tagging technique is the first that enables site-specific (11)C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, (11)C-labeled Sel-tagged Z(HER2:342) can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for 99mTc-labelling at the C terminus.

Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide (99m)Tc. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with (99m)Tc. (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, (99m)Tc-Z(HER2:2395)-VDC and (99m)Tc-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of (99m)Tc-Z(HER2:2395)-VDC, but it was substantially higher than uptake of (99m)Tc-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of (99m)Tc was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of (99m)Tc labelling due to a partial loss of site-specificity of nuclide chelation.

Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules.

Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

Molecular design and optimization of 99mTc-labeled recombinant affibody molecules improves their biodistribution and imaging properties.

UNLABELLED: Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). (99m)Tc as a label offers advantages in clinical practice, and earlier studies demonstrated that (99m)Tc-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting. METHODS: A series of recombinant derivatives of the HER2-binding Z(HER2)(:342) Affibody molecule with a C-terminal chelating sequence, -GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with (99m)Tc and evaluated in vitro and in vivo. RESULTS: All variants were stably labeled with (99m)Tc, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, (99m)Tc-Z(HER2)(:V2), with the C-terminal chelating sequence -GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. (99m)Tc-Z(HER2)(:V2) displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 ± 4.0 and 7.7 ± 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake. CONCLUSION: These results demonstrate that the biodistribution properties of recombinant (99m)Tc-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. (99m)Tc-Z(HER2)(:V2) is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.

Optimal specific radioactivity of anti-HER2 Affibody molecules enables discrimination between xenografts with high and low HER2 expression levels.

PURPOSE: Overexpression of the HER2 receptor is a biomarker for predicting those patients who may benefit from trastuzumab therapy. Radiolabelled Affibody molecules can be used to visualize HER2 expression in tumour xenografts with high sensitivity. However, previous studies demonstrated that the difference in uptake in xenografts with high and low HER2 expression levels is not proportional to the difference in expression levels. We hypothesized that discrimination between tumours with high and low HER2 expression may be improved by increasing the injected dose (reducing the specific activity) of the tracer. METHODS: The influence of injected dose of anti-HER2 (111)In-DOTA-Z(HER2 342) Affibody molecule on uptake in SKOV-3 (high HER2 expression) and LS174T (low expression) xenografts was investigated. The optimal range of injected doses enabling discrimination between xenografts with high and low expression was determined. To verify this, tumour uptake was measured in mice carrying both SKOV-3 and LS174T xenografts after injection of either 1 or 15 µg (111)In-DOTA-Z(HER2:342). RESULTS: An increase in the injected dose caused a linear decrease in the radioactivity accumulation in the LS174T xenografts (low HER2 expression). For SKOV-3 xenografts, the dependence of the tumour uptake on the injected dose was less dramatic. The injection of 10-30 µg (111)In-DOTA-Z(HER2:342) per mouse led to the largest difference in uptake between the two types of tumour. Experiments in mice bearing two xenografts confirmed that the optimized injected dose enabled better discrimination of expression levels. CONCLUSION: Careful optimization of the injected dose of Affibody molecules is required for maximum discrimination between xenografts with high and low levels of HER2 expression. This information has potential relevance for clinical imaging applications.

Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand.

Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ß-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope ((99m)Tc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.

Targeting of HER2-expressing tumors using 111In-ABY-025, a second-generation affibody molecule with a fundamentally reengineered scaffold.

UNLABELLED: Overexpression of the human epidermal growth factor receptor type 2 (HER2) in breast carcinomas predicts response to trastuzumab therapy. Affibody molecules based on a nonimmunoglobulin scaffold have demonstrated a high potential for in vivo molecular imaging of HER2-expressing tumors. The reengineering of the molecular scaffold has led to a second generation of optimized Affibody molecules that have a surface distinctly different from the parental protein domain from staphylococcal protein A. Compared with the parental molecule, the new tracer showed a further increased melting point, stability, and overall hydrophilicity and was more amenable to chemical peptide synthesis. The goal of this study was to assess the potential effects of this extensive reengineering on HER2 targeting, using ABY-025, a DOTA-conjugated variant of the novel tracer. METHODS: (111)In-ABY-025 was compared with previously evaluated parent HER2-binding Affibody tracers in vitro and in vivo. The in vivo behavior was further evaluated in mice bearing SKOV-3 xenografts, rats, and cynomolgus macaques (Macaca fascicularis). RESULTS: (111)In-ABY-025 bound specifically to HER2 in vitro and in vivo. Direct comparison with the previous generation of HER2-binding tracers showed that ABY-025 retained excellent targeting properties. Rapid blood clearance was shown in mice, rats, and macaques. A highly specific tumor uptake of 16.7 +/- 2.5 percentage injected activity per gram of tissue was seen at 4 h after injection. The tumor-to-blood ratio was 6.3 at 0.5 h and 88 at 4 h and increased up to 3 d after injection. gamma-camera imaging of tumors was already possible at 0.5 h after injection. Furthermore, the repeated intravenous administration of ABY-025 did not induce antibody formation in rats. CONCLUSION: The biodistribution of (111)In-ABY-025 was in remarkably good agreement with the parent tracers, despite profound reengineering of the nonbinding surface. The molecule displayed rapid blood clearance in all species investigated and excellent targeting capacity in tumor-bearing mice, leading to high tumor-to-organ-ratios and high-contrast imaging shortly after injection.

Evaluation of the radiocobalt-labeled [MMA-DOTA-Cys61]-Z HER2:2395(-Cys) affibody molecule for targeting of HER2-expressing tumors.

PURPOSE: Imaging using positron emission tomography (PET) in the field of nuclear medicine is becoming increasingly important. The aim of this study was to develop a method for labeling of affibody molecules with radiocobalt for PET applications. PROCEDURES: The human epidermal growth factor receptors type 2 (HER2) binding affibody molecule DOTA-Z(2395)-C was radiolabeled with (57)Co (used as a surrogate of (55)Co). The binding specificity and cellular processing of the labeled compound was studied in vitro followed by in vivo characterization in normal and tumor-bearing mice. Furthermore, a comparative biodistribution study was performed with a (111)In-labeled counterpart. RESULTS: DOTA-Z(2395)-C was successfully labeled with radiocobalt with nearly quantitative yield. The compound displayed good retention on cells over time and high tumor accumulation of radioactivity in animal studies. Imaging studies showed clear visualization of HER2-positive tumors. Furthermore, the radiocobalt label provided better tumor-to-organ ratios than (111)In. CONCLUSIONS: Radiocobalt is a promising label for affibody molecules for future PET applications.

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.

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).

Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

The influence of Bz-DOTA and CHX-A''-DTPA on the biodistribution of ABD-fused anti-HER2 Affibody molecules: implications for (114m)In-mediated targeting therapy.

PURPOSE: Affibody molecules represent a novel class of high-affinity agents for radionuclide tumour targeting. Fusion of the Affibody molecules with an albumin-binding domain (ABD) enables modification of the blood kinetics of the Affibody molecules and reduction of the renal dose. (177)Lu-CHX-A''-DTPA-ABD-(Z(HER2:342))(2), an anti-HER2 Affibody molecule-ABD fusion protein has earlier demonstrated promising results in treatment of HER2-expressing micro-xenografts in mice. The use of the in vivo generator (114m)In/(114)In as a label for ABD-fused Affibody molecules would create preconditions for efficient treatment of both micrometastases (due to conversion and Auger electrons of (114m)In) and bulky tumours (due to high-energy beta particles from the daughter nuclide (114)In). The goal of this study was to investigate if different chelators influence the biodistribution of ABD-(Z(HER2:342))(2) and to find an optimal chelator for attachment of (114m)In to the Affibody molecule-ABD fusion protein. METHODS: Isothiocyanate derivatives of Bz-DOTA and CHX-A''-DTPA were coupled to ABD-(Z(HER2:342))(2). The cellular processing of both conjugates was studied in vitro. The influence of chelators on the biodistribution was investigated in mice using double isotope ((114m)In and (111)In) labelling. RESULTS: The apparent affinity of CHX-A''-DTPA-ABD-(Z(HER2:342))(2) and Bz-DOTA-ABD-(Z(HER2:342))(2) to the extracellular domain of HER2 was similar, 13.5 and 15.0 pM, respectively. It was found that both conjugates were internalized by SKOV-3 cells. The use of CHX-A''-DTPA provided better cellular retention of the radioactivity, better tumour accumulation of radioactivity and better tumour to organ dose ratios than Bz-DOTA-ABD-(Z(HER2:342))(2). CONCLUSION: CHX-A''-DTPA is more suitable for (114m)In labelling of Affibody molecule-ABD fusion proteins for radionuclide therapy.

Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine.

UNLABELLED: The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. METHODS: A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts. RESULTS: (99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys. CONCLUSION: The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.

On the selection of a tracer for PET imaging of HER2-expressing tumors: direct comparison of a 124I-labeled affibody molecule and trastuzumab in a murine xenograft model.

UNLABELLED: Human epidermal growth factor receptor type 2 (HER2) is a tyrosine kinase, which is often overexpressed in many carcinomas. Imaging HER2 expression in malignant tumors can provide important prognostic and predictive diagnostic information. The use of anti-HER2 tracers labeled with positron-emitting radionuclides may increase the sensitivity of HER2 imaging. The goal of this study was to compare directly 2 approaches for developing anti-HER2 PET tracers: a (124)I-labeled monoclonal antibody and a small (7-kDa) scaffold protein, the Affibody molecule. METHODS: The anti-HER2 Affibody Z(HER2:342) and humanized monoclonal antibody trastuzumab were labeled with (124/125)I using p-iodobenzoate (PIB) as a linker. Cellular processing of both tracers by HER2-expressing cells was investigated. The biodistributions of (124)I-PIB-Z(HER2:342) and (125)I-PIB-trastuzumab were compared in BALB/C nu/nu mice bearing HER2-expressing NCI-N87 xenografts using paired labels. Small-animal PET of (124)I-PIB-Z(HER2:342) and (124)I-PIB-trastuzumab in tumor-bearing mice was performed at 6, 24, and 72 h after injection. RESULTS: Both radioiodinated Z(HER2:342) and trastuzumab bound specifically to HER2-expressing cells in vitro and specifically targeted HER2-expressing xenografts in vivo. Radioiodinated trastuzumab was more rapidly internalized and degraded, which resulted in better retention of radioactivity delivered by Z(HER2:342). Total uptake of trastuzumab in tumors was higher than that of (124)I-PIB-Z(HER2:342). However, tumor-to-organ ratios were appreciably higher for (124)I-PIB-Z(HER2:342) due to the more rapid clearance of radioactivity from blood and normal organs. The ex vivo results were confirmed by small-animal PET. CONCLUSION: The use of the small scaffold targeting Affibody provides better contrast in HER2 imaging than does the monoclonal antibody.

Slow internalization of anti-HER2 synthetic affibody monomer 111In-DOTA-ZHER2:342-pep2: implications for development of labeled tracers.

Affibody molecules are a novel class of targeting proteins, demonstrating promising results in the molecular imaging of tumor markers. The aim of this study was to investigate the cellular processing of Affibody molecules bound to human epidermal growth-factor-receptor type 2 (HER2). Cellular processing of the synthetic Affibody molecule, DOTA-Z(HER2:342-pep2) (K(D) = 65 (p)M) labeled with indium-111, was studied both during continuous and interrupted incubation with HER2-expressing cell lines (SKOV-3, SKBR-3, and BT474). The internalized and membrane bound fractions of Affibody molecule were discriminated by treatment with 4 M of urea solution in 0.2 M of glycine buffer (pH 2.0). Incubation media collected after an interrupted incubation was analyzed for the presence of radiocatabolites. Continuous incubation of tumor cells with (111)In-DOTA-Z(HER2:342-pep2) led to the saturation of HER2 and slow internalization. Sixty (60)- to 80% of the radioactivity remained cell associated 24 hours after interrupted incubation. The rate of Affibody molecule internalization was the same after interrupted incubation, as in the continuous incubation experiments. Internalization of (111) In-DOTA-Z(HER2:342-pep2) was relatively slow. A high level of cellular retention of the tracer was provided by strong binding to cell-surface receptors. These data suggest that good tumor targeting with anti-HER Affibody molecules may be obtained by using short-lived, nonresidualizing labels.