Novel affibody molecules as potential agents in molecular imaging for MAGE-A3-positive tumor diagnosis

The cancer-testis protein melanoma antigen A3 (MAGE-A3) is highly expressed in a broad range of malignant tumor forms. It has been confirmed that affibody molecules, a novel family of small (∼6.5 kDa) targeting proteins, are useful agents for molecular imaging and targeted tumor treatment. As a novel agent for in vivo molecular imaging detection of MAGE-A3-positive tumors, the efficacy of affibody molecules was assessed in this research. In this study, three cycles of phage display library screening resulted in the isolation of two new affibody molecules (ZMAGE-A3:172 and ZMAGE-A3:770) that attach to MAGE-A3. These molecules were then expressed in bacteria and purified. The affibody molecules with high affinity and specificity were evaluated using western blotting, immunohistochemistry, indirect immunofluorescence, surface plasmon resonance, and near-infrared optical imaging of tumor-bearing nude mice. The selected ZMAGE-A3 affibodies can precisely bind to the MAGE-A3 protein in living cells and display high-affinity binding to the MAGE-A3 protein at the molecular level. Furthermore, the accumulation of DyLight755-labeled ZMAGE-A3:172 or ZMAGE-A3:770 in MAGE-A3-positive tumors was achieved as early as 30 min and disappeared at 48 h post-injection. Our findings support the potential of the two MAGE-A3 protein-binding affibody molecules for their use as molecular imaging agents. •Affibody molecules effectively detected MAGE-A3-positive tumors in vivo for molecular imaging.•Co-localization and co-immunoprecipitation assays confirmed affibodies' high specificity for binding to natural MAGE-A3 protein in cells.•Analysis of tumor imaging in xenograft mice model confirmed specific accumulation of two novel ZMAGE-A3 affibody molecules at tumor site and kidney, effectively locating MAGE-A3 protein in cancer cells in vivo.•Ex vivo imaging of tumor tissue and mouse organs revealed strong fluorescence signal, suggesting accumulation of two DyLight755-labeled ZMAGE-A3 affibody molecules in tumor tissue from A375 and SGC-7901 xenograft mice model, supporting in vivo molecular imaging results.•These two novel affibody molecules exhibit high affinity and specificity for MAGE-A3 protein in MAGE-A3-positive cancer cells.