The family of vascular endothelial growth factor (VEGF) ligands and their interactions with VEGF receptors (VEGFRs) play important roles in both pathological and physiological angiogenesis. Hence, agonistic and antagonistic ligands targeting this signaling pathway have potential for both studies on fundamental biology and for development of therapies and diagnostics. Here, we engineer VEGFR2-binding affibody molecules for increased thermostability, refolding and improved biodistribution. We designed libraries based on the original monomeric binders with the intention of reducing hydrophobicity, while retaining high affinity for VEGFR2. Libraries were displayed on bacteria and binders were isolated by fluorescence-activated cell sorting (FACS). In parallel, we used an automated sequence- and structure-based in silico algorithm to identify potentially stabilizing mutations. Monomeric variants isolated from the screening and the in silico approach, respectively, were characterized by circular dichroism spectroscopy and biosensor assays. The most promising mutations were combined into new monomeric constructs which were finally fused into a dimeric construct, resulting in a 15 degrees C increase in melting temperature, complete refolding capability after heat-induced denaturation, retained low picomolar affinity and improved biodistribution profile in an in vivo mouse model. These VEGFR2-binding affibody molecules show promise as candidates for further in vivo studies to assess their suitability as molecular imaging and therapeutic agents.

Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9-29-H 6 and G3-H 6 were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H 6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H 6 in comparison to 9-29-H 6 . Technetium-99m labeled G3-H 6 demonstrated a better biodistribution profile than 9-29-H 6 , with several-fold lower uptake in liver. Radioiodinated G3-H 6 showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H 6 with high clinical potential for imaging of HER2.

Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gastroesophageal cancer is used for the stratification of patients for HER2-targeting therapies. The use of radionuclide molecular imaging may facilitate such evaluation in a non-invasive way. Designed ankyrin repeat proteins (DARPins) are engineered scaffold proteins with high potential as probes for radionuclide molecular imaging. DARPin G3 binds with high affinity to HER2 and may be used to visualize this important therapeutic target. Studies on other engineered scaffold proteins have demonstrated that selection of the optimal labeling approach improves the sensitivity and specificity of radionuclide imaging. The present study compared two methods of labeling G3, direct and indirect radioiodination, to select an approach providing the best imaging contrast. G3-H6 was labeled with iodine-124, iodine-125 and iodine-131 using a direct method. A novel construct bearing a C-terminal cysteine, G3-GGGC, was site-specifically labeled using [125I]I-iodo-[(4-hydroxyphenyl)ethyl]maleimide (HPEM). The two radiolabeled G3 variants preserved binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was demonstrated. Biodistribution comparison of [131I]I-G3-H6 and [125I]I-HPEM-G3-GGGC in mice, bearing HER2-expressing SKOV3 xenografts, demonstrated an appreciable contribution of hepatobiliary excretion to the clearance of [125I]I-HPEM-G3-GGGC and a decreased tumor uptake compared to [131I]I-G3-H6. The direct label provided higher tumor-to-blood and tumor-to-organ ratios compared with the indirect label at 4 h post-injection. The feasibility of high contrast PET/CT imaging of HER2 expression in SKOV3 xenografts in mice using [124I]I-G3-H6 was demonstrated. In conclusion, direct radioiodination is the preferable approach for labeling DARPin G3 with iodine-123 and iodine-124 for clinical single photon emission computed tomography and positron emission tomography imaging.

Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARP in G3, a small (14 kDa) engineered scaffold protein, is a promising probe for imaging of HER2. We hypothesized that position (C- or N-terminus) and composition (hexahistidine or (HE)(3)) of histidine-containing tags would influence the biodistribution of [Tc-99m]Tc(CO)(3)-labeled DARP in G3. To test the hypothesis, G3 variants containing tags at N-terminus (H-6-G3 and (HE)(3)-G3) or at C-terminus (G3-H-6 and G3-(HE)(3)) were labeled with [Tc-99m]Tc(CO)(3). Labeling yield, label stability, specificity and affinity of the binding to HER2, biodistribution and tumor targeting properties of these variants were compared side-by-side. There was no substantial influence of position and composition of the tags on binding of [Tc-99m]Tc(CO)(3)-labeled variants to HER2. The specificity of HER2 targeting in vivo was confirmed. The tumor uptake in BALB/c nu/nu mice bearing SKOV3 xenografts was similar for all variants. On the opposite, there was a strong influence of the tags on uptake in normal tissues. The tumor-to-liver ratio for [Tc-99m]Tc(CO)(3)-(HE)(3)-G3 was three-fold higher compared to the hexahistidine-tag containing variants. Overall, [Tc-99m]Tc(CO)(3)-(HE)(3)-G3 variant provided the highest tumor-to-lung, tumor-to-liver, tumor-to-bone and tumor-to-muscle ratios, which should improve sensitivity of HER2 imaging in these common metastatic sites.

Strategies to promote vascularization are being developed in order to improve long-term survival of artificial tissue constructs. Vascular endothelial growth factor A (VEGFA) has an important role in both pathological and physiological angiogenesis, mediated by binding to VEGF receptors (VEGFRs). In nature, signaling can be modulated by presentation of growth factors in either soluble form or bound to the extracellular matrix. Herein, a previously reported VEGFR2-binding antagonistic affibody heterodimer (di-Z(VEGFR2)) was formatted into a tetrameric construct (tetra-Z(VEGFR2)) with the intention of generating artificial agonistic ligands for VEGFR2 signaling. In vitro cell assays demonstrated that tetra-Z(VEGFR2) induced VEGFR2 phosphorylation and increased cell proliferation, in contrast to di-Z(VEGFR2). In order to simulate matrix-bound factors, both constructs were fused at the genetic level to a partial spider silk protein, 4RepCT. Assembly of the silk fusion proteins onto a solid surface was verified by quartz crystal microbalance with dissipation analysis. Moreover, surface plasmon resonance studies demonstrated retained VEGFR2 binding ability of di-Z(VEGFR2) silk and tetra-Z(VEGFR2)-silk after silk-mediated immobilization. Cell culture studies demonstrated that VEGFR2-overexpressing cells adhered to di-Z(VEGFR2) -silk and tetra-Z(VEGFR2) -silk and had activated VEGFR2 signaling. Altogether, we demonstrate the potential of especially tetra-Z(VEGFR2)-silk to promote angiogenesis in tissue-engineering applications. The results from the study also show that molecules can obtain completely new functions when presented on materials, and verifying the biological effects after functionalizing materials is thus always recommended.

Vascular endothelial growth factor receptor-2 (VEGFR2) is a key mediator of angiogenesis and therefore a promising therapeutic target in malignancies including glioblastoma multiforme (GBM). Molecular imaging of VEGFR2 expression may enable patient stratification for antiangiogenic therapy. The goal of the current study was to evaluate the capacity of the novel anti-VEGFR2 biparatopic affibody conjugate (Z(VEGFR2)-Bp(2)) for in vivo visualization of VEGFR2 expression in GBM. Methods: Z(VEGFR2)-Bp(2) coupled to a NODAGA chelator was generated and radiolabeled with indium-111. The VEGFR2-expressing murine endothelial cell line MS1 was used to evaluate in vitro binding specificity and affinity, cellular processing and targeting specificity in mice. Further tumor targeting was studied in vivo in GL261 glioblastoma orthotopic tumors. Experimental imaging was performed. Results: [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) bound specifically to VEGFR2 (K-D=33 +/- 18 pM). VEGFR2-mediated accumulation was observed in liver, spleen and lungs. The tumor-to-organ ratios 2 h post injection for mice bearing MS1 tumors were approximately 11 for blood, 15 for muscles and 78 for brain. Intracranial GL261 glioblastoma was visualized using SPECT/CT. The activity uptake in tumors was significantly higher than in normal brain tissue. The tumor-to-cerebellum ratios after injection of 4 mu g [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) were significantly higher than the ratios observed for the 40 mu g injected dose and for the non-VEGFR2 binding size-matched conjugate, demonstrating target specificity. Microautoradiography of cryosectioned CNS tissue was in good agreement with the SPECT/CT images. Conclusion: The anti-VEGFR2 affibody conjugate [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) specifically targeted VEGFR2 in vivo and visualized its expression in a murine GBM orthotopic model. Tumor-to-blood ratios for [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) were higher compared to other VEGFR2 imaging probes. [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) appears to be a promising probe for in vivo noninvasive visualization of tumor angiogenesis in glioblastoma.