In vivo targeting of HER2-positive tumor using 2-helix affibody molecules.

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, 68Ga and 18F, with relatively short half-life (t1/2<2 h).

64Cu-labeled affibody molecules for imaging of HER2 expressing tumors.

Introduction: The development of molecular probes based on novel engineered protein constructs is under active investigation due to the great potential of this generalizable strategy for imaging a variety of tumor targets.

Discussion: In this report, human epidermal growth factor receptor type 2 (HER2)-binding Affibody molecules were radiolabeled with (64)Cu and their imaging ability was further evaluated in tumor mice models to understand the promise and limitations of such probes. The anti-HER2 Affibody molecules in monomeric (Z(HER2:477)) and dimeric [(Z(HER2:477))(2)] forms were site specifically modified with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetate mono (N-ethylmaleimide amide) (Mal-DOTA).

A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression.

Unlabelled: Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix-based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one alpha-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2).

Engineered two-helix small proteins for molecular recognition.

Less is more: By starting with a high-affinity HER2-binding 3-helix affibody molecule, we successfully developed 2-helix small protein binders with 5 nM affinities by using a combination of several different strategies. Our efforts clearly suggest that 2-helix small proteins against important tumor targets can be obtained by rational protein design and engineering.

A novel method for direct site-specific radiolabeling of peptides using [18F]FDG.

We have used the well-accepted and easily available 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) positron emission tomography (PET) tracer as a prosthetic group for synthesis of (18)F-labeled peptides. We herein report the synthesis of [(18)F]FDG-RGD ((18)F labeled linear RGD) and [(18)F]FDG-cyclo(RGD(D)YK) ((18)F labeled cyclic RGD) as examples of the use of [(18)F]FDG. We have successfully prepared [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) in 27.

Direct site-specific radiolabeling of an Affibody protein with 4-[18F]fluorobenzaldehyde via oxime chemistry.

Purpose: In this study, we introduce a methodology for preparing 18F-labeled Affibody protein, specifically 18F-Anti-HER2 dimeric Affibody (14 kDa), for in vivo imaging of HER2neu with positron emission tomography (PET).

Procedures: We have used 4-[18F]fluorobenzaldehyde as a synthon to prepare 18F-Anti-HER2 Affibody. Aminooxy-functionalized Affibody (Anti-HER2-ONH2) was incubated with 4-[18F]fluorobenzaldehyde in ammonium acetate buffer at pH 4 in the presence of methanol at 70 degrees C for 15 min.

Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules.

Unlabelled: Human epidermal growth factor receptor type 2 (HER2) is a well-established tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of (18)F-labeled biomolecules for PET imaging of HER2 (HER2 PET) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment.

An autonomously folding beta-hairpin derived from the human YAP65 WW domain: attempts to define a minimum ligand-binding motif.

WW domains are broadly distributed among natural proteins; these modules play a role in bringing specific proteins together. The ligands recognized by WW domains are short segments rich in proline residues. We have tried to identify the minimum substructure within a WW domain that is required for ligand binding.

Influence of strand number on antiparallel beta-sheet stability in designed three- and four-stranded beta-sheets.

We describe experiments that probe whether antiparallel beta-sheet secondary structure becomes more stable as the number of strands increases. Several groups, including ours, have explored this issue with peptides designed to adopt three-stranded beta-sheet conformations, but the conclusions have not been consistent. In this study, we examine the effect on conformational stability of beta-sheet lengthening perpendicular to the strand direction via analysis of designed peptides that adopt three-stranded or four-stranded antiparallel beta-sheet conformations in aqueous solution.

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet.

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1.

Spectroscopic characterization of selected beta- sheet hairpin models.

The IR and vibrational circular dichroism (VCD) spectra of a model two-stranded beta hairpin are compared to those of a related cyclic two-stranded model, which are both stabilized by DPro- Gly turns. The spectra are compared to ab initio based simulations to support specific assignments of the dominant features and suggest a revised interpretation of the IR and VCD spectra for beta sheet containing proteins.