Camilla Hofström

HEHEHE-Tagged Affibody Molecule May Be Purified by IMAC, Is Conveniently Labeled with [99mTc(CO)3]+, and Shows Improved Biodistribution with Reduced Hepatic Radioactivity Accumulation

Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [⁹⁹(m)Tc(CO)₃](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER)₂(:)₃₄₂ were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [⁹⁹(m)Tc(CO)₃](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER)₂(:)₃₄₂-H₆ and (HE)₃-Z(HER)₂(:)₃₄₂, respectively, could be efficiently purified using IMAC and stably labeled with [⁹⁹(m)Tc(CO)₃](+) and were subsequently compared with the parental H₆-Z(HER)₂(:)₃₄₂ having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [⁹⁹(m)Tc(CO)₃](+)-Z(HER2:342)-H₆ compared to [⁹⁹(m)Tc(CO)₃](+)-H₆-Z(HER)₂(:)₃₄₂, and more than 10-fold lower with [⁹⁹(m)Tc(CO)₃](+)-(HE)₃-Z(HER)₂(:)₃₄₂. These differences translated into appreciably superior tumor-to-liver ratio for [⁹⁹(m)Tc(CO)₃](+)-(HE)₃-Z(HER)₂(:)₃₄₂ compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.

Imaging of Insulinlike Growth Factor Type 1 Receptor in Prostate Cancer Xenografts Using the Affibody Molecule 111In-DOTA-ZIGF1R:4551

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 ZIGF1R:4551 Affibody molecule was site-specifically conjugated with a maleimido derivative of DOTA and labeled with 111In. The binding of radiolabeled ZIGF1R:4551 to IGF-1R–expressing cells was evaluated in vitro and in vivo. Results: DOTA-ZIGF1R:4551 can be stably labeled with 111In with preserved specific binding to IGF-1R–expressing cells in vitro. In mice, 111In-DOTA-ZIGF1R: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.

Use of a HEHEHE Purification Tag Instead of a Hexahistidine Tag Improves Biodistribution of Affibody Molecules Site-Specifically Labeled with Tc-99m, In-111 and I-125

Affibody molecules are a class of small (∼7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging in vivo. The attachment of a hexahistidine (His(6))-tag to the Affibody molecule allows facile purification by immobilized metal ion affinity chromatography (IMAC) but leads to high accumulation of radioactivity in the liver. Earlier, we have 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 IMAC, enables labeling with [(99m)Tc(CO)(3)](+), and provides low hepatic accumulation of radioactivity. In this study, we compared the biodistribution of cysteine-containing Affibody molecules site-specifically labeled with (111)In, (99m)Tc, and (125)I at the C-terminus, having a His(6)-tag at the N- or C-terminus or a HEHEHE-tag at the N-terminus. We show that the use of a HEHEHE-tag provides appreciable reduction of hepatic radioactivity, especially for radiometal labels. We hope that this information can also be useful for development of other scaffold protein-based imaging agents.

HAHAHA, HEHEHE,HIHIHI or HKHKHK : influence of position and composition of histidine containing tags on biodistribution of [99mTc(CO)3]+-labeled Affibody molecules

Engineered affibody molecules can be used for high contrast in vivo molecular imaging. Extending a recombinantly produced HER2 binding affibody molecule with a hexa-histidine tag allows for convenient purification by immobilized metal-ion affinity chromatography and labeling with [(99m)Tc(CO)3](+) but increases radioactivity uptake in the liver. To investigate the impact of charge, lipophilicity, and position on biodistribution, 10 variants of a histidine-based tag was attached to a HER2 binding affibody molecule. The biochemical properties and the HER2 binding affinity appeared to be similar for all variants. In vivo, positive charge promoted liver uptake. For N-terminally placed tags, lipophilicity promoted liver uptake and decreased kidney uptake. Kidney uptake was higher for C-terminally placed tags compared to their N-terminal counterparts. The variant with the amino acid composition HEHEHE placed in the N-terminus gave the lowest nonspecific uptake.

Novel antigen design for the generation of antibodies to G-protein-coupled receptors

Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.