No abstract

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.

Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARPin 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 [ 99m Tc]Tc(CO) 3-labeled DARPin 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 [ 99m Tc]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 [ 99m Tc]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 [ 99m Tc]Tc(CO) 3-(HE) 3-G3 was threefold higher compared to the hexahistidine-tag containing variants. overall, [ 99m Tc]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.

Radionuclide molecular imaging of human epidermal growth factor type 2 (HER2) expression may enable a non-invasive discrimination between HER2-positive and HER2-negative breast cancers for stratification of patients for HER2-targeted treatments. DARPin G3 is a small (molecular weigh 14 kDa) scaffold protein with picomolar affinity to HER2. The aim of this firstin-human study was to evaluate the safety, biodistribution and dosimetry of 99m Tc-(HE)3-G3.Methods. Three cohorts of patients with primary breast cancer (each including at least 4 patients with HER2-negative and 5 patients with HER2-positive tumors) were injected with either 1000, 2000 or 3000 µg of 99m Tc-(HE)3-G3 (287±170 MBq). Whole-body planar imaging followed by SPECT was performed at 2, 4, 6 and 24 h after injection. Vital signs and possible side effects were monitored during imaging and up to 7 days after injection.Results. All injections were well tolerated. No side effects were observed. The results of blood and urine analyses did not differ before and after studies. 99m Tc-(HE)3-G3 cleared rapidly from the blood. The highest uptake was detected in the kidneys and liver followed by the lungs, breasts and small intestinal content. The hepatic uptake after injecting with 2000 or 3000 µg was significantly (p<0.05) lower than the uptake after injecting with 1000 µg. Effective doses did not differ significantly between cohorts (average 0.011± 0.004 mSv/MBq). Tumor-to-contralateral site ratios for HER-positive tumors were significantly (p< 0.05) higher than for HER2-negative at 2 and 4 h after injection. Conclusions.Imaging of HER2 expression using 99m Tc-(HE)3-G3 is safe, well-tolerated and provides a low absorbed dose burden on patients. This imaging enables discerning HER2-positive and HER2-negative breast cancer. Phase I study data justifies further clinical development of 99m Tc-(HE)3-G3.

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.