Molecular Imaging of HER2 Expression using Synthetic Affibody Molecules Design, Synthesis and Biological Evaluation

Abstract: Molecular imaging is an emerging multidisciplinary field that addresses the visualisation of diseases at the cellular and molecular levels. This thesis focuses on the development of a synthetic Affibody molecule-based imaging tracer for the detection of HER2 expression in malignant tumours.Papers I-IV report the development of the HER2-specific Affibody molecule, ZHER2:342 by peptide synthesis and the use of different chelators attached to the N-terminus to allow 99mTc-labelling. Paper I described the optimisation of labelling of Affibody molecules using cysteine-based chelator sequences, in which the direct labelling method under alkaline conditions was the most suitable one. Papers II-IV report the development and optimisation of the in vivo properties of the HER2-specific Affibody molecule for high-contrast imaging. By using an array of mercaptoacetyl-based chelators, it was found that the substitution of a single amino acid in a 60 amino acid-long Affibody molecule can dramatically change the pharmacokinetics of the tracer. Strategic approaches that utilised hydrophilic amino acids, such as serine, glutamate and lysine, changed the excretion pathway from hepatobiliary to renal excretion. Problems with the high accumulation of radioactivity in the abdomen area and restricted imaging were resolved by the use of mercaptoacetyl-triglutamyl, maEEE or mercaptoacetyl-seryl-lysyl-seryl, maSKS chelators.Paper V reports the re-engineering of the HER2-specific Affibody molecule to provide a C-terminal SECG sequence using peptide synthesis. Incorporation of this sequence provided a multifunctional platform for labelling (with technetium or trivalent radiometals) and a flexible production route (recombinant or chemical synthesis). Combination of a serine, a glutamic acid and a thiol-bearing group in the chelating sequence reduced the renal accumulation of Affibody molecules.Altogether, the in vivo efficiency of Affibody molecules to target tumours and their biodistribution properties can be improved by strategic design and suitable chemistry. Hopefully, these observations will be applicable to other small peptide and protein scaffold-based tracers.